Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.Designing targeted therapy for cancer has been a major goal of the last decade. Oncogenic tyrosine kinases have raised early interest, because elucidation of their structure facilitated the development of small-molecule inhibitors with therapeutic efficiency.¹ The pioneer BCR-ABL inhibitor molecule imatinib was approved for therapeutic use as early as 2001 to treat chronic myeloid leukemia and Ph1-positive acute lymphoblastic leukemia.² Later on, inhibitors targeting receptors for epidermal growth factor or vascular endothelial growth factor were approved for treatment of solid tumors, such as lung and breast cancer. To date, many tyrosine kinase inhibitors (TKIs) are used in the clinic.³ However, cancers treated by TKIs invariably become resistant to therapy and relapse. Acquired resistance develops through various mechanisms including secondary mutations of the targeted oncogene or activation of alternative proliferative signaling pathways.⁴ It seems thus necessary to invent new strategies designed to attack the tumor on multiple fronts.ALK (anaplastic lymphoma kinase) is an oncogenic receptor tyrosine kinase associated with many tumor types. ALK was first identified in 1994 as a rearranged gene fusion (NPM–ALK) resulting from the t(2;5)(p23;q35) translocation occurring in 75% human anaplastic large-cell lymphomas (ALCLs).^{5, 6} Other translocations or gene inversions involving ALK were later described in solid tumors including 50–60% inflammatory myofibroblastic tumors, and a small proportion of diffuse large B-cell lymphomas, breast and renal carcinomas.^{7, 8} Recently, 4–8% non-small-cell lung cancer (NSCLC) were found to harbor an echinoderm microtubule-associated protein-like 4 (EML4)–ALK fusion.^{7, 9} Resulting fusion proteins associate the N-terminal portion of a protein partner (containing in most cases a dimerization domain) to the entire intracellular portion of ALK, including its tyrosine kinase domain. Subsequent dimerization of this fusion protein leads to constitutive activation of ALK kinase, resulting in enhanced signaling for cell proliferation, survival and oncogenicity.¹⁰The full-length ALK receptor cDNA codes for a transmembrane receptor tyrosine kinase of the insulin receptor superfamily, which is essentially expressed in the developing nervous system.^{11, 12} Some authors proposed the two heparin-binding factors pleiotrophin (PTN) and midkine as ligands for ALK.¹⁰ However, their binding to ALK is controversed and might be indirectly mediated by heparin.¹³ ALK kinase signaling most likely involves co-receptors and/or co-signaling molecules such as the transmembrane receptor tyrosine phosphatase beta/zeta (RPTPb/z), a receptor for PTN and midkine. In the absence of ligand, RPTPb/z dephosphorylates ALK, whereas PTN and midkine direct binding to RPTPb/z inactivates its phosphatase activity.¹⁴ Expression of the full-length ALK receptor was also observed in neuroblastoma, a pediatric tumor derived from the neural crest affecting the peripheral nervous system. The ALK kinase in neuroblastoma is most often constitutively active as a result of gain-of-function mutations or protein overexpression, due to ALK gene amplification or copy number increase.^{10, 15}ALK appears therefore as an interesting therapeutic target to treat ALK-positive tumors. Indeed, since the identification of NPM–ALK and other ALK fusions as oncogenes for ALCL and inflammatory myofibroblastic tumors,^{6, 16, 17} several pharmaceutical companies developed ALK-specific TKIs. In 2010, a TKI targeting ALK and c-MET, crizotinib¹⁸ (also called PF-02341066), was authorized in clinical trials as a second-line therapy for advanced stage NSCLC harboring EML4–ALK. The initial clinical responses were so encouraging that crizotinib is currently tested in a growing number of advanced ALK-positive tumors (clinicaltrials.gov). Nevertheless, the tumors invariably develop resistance to the inhibitor, mostly through mutations of the kinase active site.^{19, 20} Therefore, it appears necessary to design alternate treatments or to associate TKIs with other molecules. One promising strategy would be to impair distinct functions of the oncogenic tyrosine kinase through targeting different sites of the ALK protein.We recently demonstrated that the ALK receptor tyrosine kinase belongs to the functional family of so-called ‘dependence receptors''.^{21, 22} Such dependence receptors function with a dual signaling: in the presence of ligand (or a situation mimicking a ligand, e.g., inducing receptor dimerization and activation), the receptor exerts a prosurvival/antiapoptotic effect on the cell; in contrast, in absence of ligand and when the cell is submitted to environmental or genotoxic stress, a dependence receptor becomes proapoptotic. The proapoptotic effect is mediated by caspase-dependent cleavage of the receptor, either releasing or exposing a proapoptotic domain/sequence (called ‘addiction/dependence domain'' or ADD), thus amplifying the apoptotic process.²³ Molecular analysis of ALK deletion mutants allowed us to map the ADD domain of ALK to a 36-amino-acid (aa) stretch located within the juxtamembrane intracytoplasmic region of ALK. The ADD of ALK lacks homology with any known protein motif implicated in apoptotic processes and is necessary for ALK proapoptotic function.²² The purpose of the present study was to design a novel targeted therapy, taking advantage of the proapoptotic function of ALK.Our hypothesis was that a synthetic peptide could mimic the proapoptotic function of ALK. Therefore, we synthesized several peptides whose sequence reproduced the entire ADD domain (36 aa) of ALK or part of it (12 aa) to assay their effects on various tumor cell lines. We show that several of these ALK-derived peptides are proapoptotic for ALK-expressing, but not ALK-negative, tumor cells. In addition, the ALK-derived 36-aa peptide (P36) enhanced the cytotoxic effect of the ALK kinase inhibitor crizotinib in ALK-positive ALCL and neuroblastoma cell lines. Thus our results uncover a new strategy for targeting ALK-expressing tumors.     

Deeper in the human cornea proteome using nanoLC-Orbitrap MS/MS: An improvement for future studies on cornea homeostasis and pathophysiology

The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Capitalizing on multi-element interactions through bal-anced nutrition——A pathway to improve nitrogen use efficiency in China,India and North America

A viable option for increasing nitrogen (N) use efficiency and mitigation of negative impacts of N on the environment is to capitalize on multi-element interactions through implementation of nutrient management programs that provide balanced nutrition. Numerous studies have demonstrated the immediate efficacy of this approach in the developing regions like China and India as well as developed countries in North America. Based on 241 site-years of experiments in these countries, the first-year N recovery efficiency (RE) for the conventional or check treatments averaged 21% while the balanced treatments averaged 54% RE, for an average increase of 33% in RE due to balanced nutrition. Effective policies to promote adoption are most likely those that enable site-specific approaches to nutrient management decisions rather than sweeping, nation-wide incentives supporting one nutrient over another. Local farmers, advisers and officials need to be empowered with tools and information to help them define necessary changes in practices to create more balanced nutrient management.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Toad radiation reveals into-India dispersal as a source of endemism in the Western Ghats-Sri Lanka biodiversity hotspot

Background  

High taxonomic level endemism in the Western Ghats-Sri Lanka biodiversity hotspot has been typically attributed to the subcontinent's geological history of long-term isolation. Subsequent out of – and into India dispersal of species after accretion to the Eurasian mainland is therefore often seen as a biogeographic factor that 'diluted' the composition of previously isolated Indian biota. However, few molecular studies have focussed on into-India dispersal as a possible source of endemism on the subcontinent. Using c. 6000 base pairs of mitochondrial and nuclear DNA, we investigated the evolutionary history and biogeography of true toads (Bufonidae), a group that colonized the Indian Subcontinent after the Indo-Asia collision.     

Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (approximately 0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity.     

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?

In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.