Protein kinase FgSch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum

Saccharomyces cerevisiae protein kinase Sch9 is one of the downstream effectors of the target of rapamycin (TOR) complex 1 and plays multiple roles in stress resistance, longevity and nutrient sensing. However, the functions of Sch9 orthologs in filamentous fungi, particularly in pathogenic species, have not been characterized to date. Here, we investigated biological and genetic functions of FgSch9 in Fusarium graminearum. The FgSCH9 deletion mutant (ΔFgSch9) was defective in aerial hyphal growth, hyphal branching and conidial germination. The mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall‐damaging agents, and to rapamycin, while showing increased thermal tolerance. We identified FgMaf1 as one of the FgSch9‐interacting proteins that plays an important role in regulating mycotoxin biosynthesis and virulence of F. graminearum. Co‐immunoprecipitation and affinity capture‐mass spectrometry assays showed that FgSch9 also interacts with FgTor and FgHog1. More importantly, both ΔFgSch9 and FgHog1 null mutant (ΔFgHog1) exhibited increased sensitivity to osmotic and oxidative stresses. This defect was more severe in the FgSch9/FgHog1 double mutant. Taken together, we propose that FgSch9 serves as a mediator of the TOR and high osmolarity glycerol pathways, and regulates vegetative differentiation, multiple stress responses and secondary metabolism in F. graminearum.     

Endothelial Nitric Oxide Synthase (eNOS) gene polymorphisms in spontaneously aborted embryos

Endothelium-derived nitric oxide (NO) is synthesized from L-arginine by endothelial nitric oxide synthase (eNOS) encoded by the eNOS gene on chromosome 7. The effects of the eNOS polymorphisms with the risk of spontaneous pregnancy losses are conflicting. In this study, we investigated the association of the eNOS genotypes with spontaneously aborted embryos in Koreans. Case-control studies were performed to evaluate the association between endothelial nitric oxide synthase (eNOS) gene polymorphisms and spontaneously aborted embryos. Ninety-nine spontaneously aborted fetuses at <20 weeks of gestational age and 103 child controls and 282 adult controls. Genotype frequency of three eNOS gene polymorphisms, ?786T>C, VNTR in intron 4 (4a4b), and 894G>T in spontaneously aborted embryos was surveyed. The frequencies of ?786TC and CC genotypes in aborted embryos were significantly higher than in both child and adult controls. The frequencies of 4a4a homozygote of VNTR polymorphism in intron 4 and TT homozygote of 894G>T polymorphisms were also higher in aborted embryos than in adult controls. Haploptype analysis suggested that ?786T>C polymorphism was a possible risk factor for spontaneously aborted embryos. eNOS gene polymorphisms, ?786T>C, VNTR in intron 4 (4a4b), and 894G>T, are associated with the risk of spontaneously aborted fetuses.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

Development of uniform density control with self-assembled colloidal gold nanoparticles on a modified silicon substrate

Here, we present a simple method for controlling the density of Au nanoparticles (Au NPs) on a modified silicon substrate, by destabilizing the colloidal Au NPs with 3-mercaptopropyltrimethoxylsilane (3-MPTMS) for microelectromechanical-system-based applications to reduce tribological issues. A silicon surface was pretreated with a 3-MPTMS solution, immediately after which thiolated Au NPs were added to it, resulting in their uniform deposition on the silicon substrate. Without any material property change of the colloidal Au NPs, we observed the formation of large clusters Au NPs on the modified silicon surface. Analysis by scanning electron microscopy with energy dispersive X-ray spectroscopy indicated that the addition of 3-MPTMS resulted in an alternation of the chemical characteristics of the solution. Atomic force microscopy imaging supported the notion that silicon surface modification is the most important factor on tribological properties of materials along with ligand-modified Au NPs. The density of Au NPs on a silicon surface was significantly dependent on several factors, including the concentration of colloidal Au NPs, deposition time, and concentration of 3-MPTMS solution, while temperature range which was used throughout experiment was determined to have no significant effect. A relatively high density of Au NPs forms on the silicon surface as the concentrations of Au NPs and 3-MPTMS are increased. In addition, the maximum deposition of Au NPs on silicon wafer was observed at 3 h, while the effects of temperature variation were minimal.     

An In Vivo C. elegans Model System for Screening EGFR-Inhibiting Anti-Cancer Drugs

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.     

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ: IDENTIFICATION OF KEY CB1 CONTACTS WITH THE C-TERMINAL HELIX α5 OF Gαi*

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and G_i heterotrimer (CB1-G_i), guided by the x-ray structure of β₂-adrenergic receptor (β₂AR) in complex with G_s (β₂AR-G_s), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218^3.54, Tyr-224^IC2, Asp-338^6.30, Arg-340^6.32, Leu-341^6.33, and Thr-344^6.36, as potential key contacts with the extreme C-terminal helix α₅ of Gα_i. Ala mutations of these residues at the receptor-G_i interface resulted in little G protein coupling activity, consistent with the present model of the CB1-G_i complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α₅ of Gα_i. The model also suggests that unique conformational changes in the extreme C-terminal helix α₅ of Gα play a crucial role in the receptor-mediated G protein activation.     

Aldose reductase inhibitory compounds from extracts of Dipsacus asper

Amethanolic extract of Dipsacus asper, having anti-diabetic activity, was examined as a possible aldose reductase (ALR2) inhibitor, a key enzyme involved in diabetic complications. Bioactivity guided fractionation led to the isolation of ten compounds, ursolic acid (1), oleanolic acid-3-O-α-L-arabinopyranoside (2), daucosterol (3), hederagenin-3-O-α-L-arabinopyranoside (4), sweroside(5), caffeic acid (6), esculetin (7), protocatechualdehyde (8), loganin (9), and vanilic acid (10) from the ethyl acetate fraction of D. asper methanol extract. Among them, compounds 4, 6, 7, and 8 exhibited inhibitory effects on aldose reductase, with IC₅₀ values of 23.70, 16.71, 34.36, and 21.81 μM, respectively. This is the first report on the isolation of these compounds from D. asper, and the ALR2 inhibitory activity of hederagenin-3-O-α-L-arabinopyranoside. These results suggest the successful use of the extract of D. asper for ameliorating diabetic complications.     

The Colletotrichum graminicola striatin orthologue Str1 is necessary for anastomosis and is a virulence factor

Striatin family proteins are key regulators in signalling pathways in fungi and animals. These scaffold proteins contain four conserved domains: a caveolin‐binding domain, a coiled‐coil motif and a calmodulin‐binding domain at the N‐terminus, and a WD‐repeat domain at the C‐terminus. Fungal striatin orthologues are associated with sexual development, hyphal growth and plant pathogenesis. In Fusarium verticillioides, the striatin orthologue Fsr1 promotes virulence in the maize stalk. The relationship between fungal striatins and pathogenicity remains largely unexplored. In this study, we demonstrate that the Colletotrichum graminicola striatin orthologue Str1 is required for full stalk rot and leaf blight virulence in maize. Pathogenicity assays show that the striatin mutant strain (Δstr1) produces functional appressoria, but infection and colonization are attenuated. Additional phenotypes of the Δstr1 mutant include reduced radial growth and compromised hyphal fusion. In comparison with the wild‐type, Δstr1 also shows a defect in sexual development and produces fewer and shorter conidia. Together with the fact that F. verticillioides fsr1 can complement Δstr1, our results indicate that C. graminicola Str1 shares five phenotypes with striatin orthologues in other fungal species: hyphal growth, hyphal fusion, conidiation, sexual development and virulence. We propose that fungal striatins, like mammalian striatins, act as scaffolding molecules that cross‐link multiple signal transduction pathways.     

Elucidation of antifungal metabolites produced by Pseudomonas aurantiaca IB5-10 with broad-spectrum antifungal activity

Antifungal metabolites were isolated from a culture of Pseudomonas aurantiaca IB5-10. Chemical structures of the metabolites were elucidated as phenazine-1-carboxylic acid (PCA; 1), 2-hydroxyphenazine (2-OH-PHZ; 2), and cyclo-(L-Pro-L-Val; 3), respectively, based on spectroscopic methods. Among them, 3 was isolated for the first time from this strain. The antifungal activities of 1-3 were evaluated against a variety of plant pathogens. To the best of our knowledge, the antifungal activities of 3 against plant fungal pathogens have been evaluated for the first time in this work. PCA (1) showed the most potent antifungal activities against Phytophthora capsici, Rhizoctonia solani AG-1(IA), and Pythium ultimum with MICs (microgram/ml) of less than 1.0, 1.3, and 2.0, respectively. On the other hand, 2-OH-PHZ (2) showed potent antifungal activity against R. solani AG-1(IA) with the MIC (microgram/ml) of 2.0, whereas it showed moderate antifungal activity against P. ultimum with the MIC (microgram/ml) of 50.0. In addition, 3 showed antifungal activity against only R. solani AG- 1(IA).     

The use of sparse CT datasets for auto-generating accurate FE models of the femur and pelvis

The finite element (FE) method when coupled with computed tomography (CT) is a powerful tool in orthopaedic biomechanics. However, substantial data is required for patient-specific modelling. Here we present a new method for generating a FE model with a minimum amount of patient data. Our method uses high order cubic Hermite basis functions for mesh generation and least-square fits the mesh to the dataset. We have tested our method on seven patient data sets obtained from CT assisted osteodensitometry of the proximal femur. Using only 12 CT slices we generated smooth and accurate meshes of the proximal femur with a geometric root mean square (RMS) error of less than 1 mm and peak errors less than 8 mm. To model the complex geometry of the pelvis we developed a hybrid method which supplements sparse patient data with data from the visible human data set. We tested this method on three patient data sets, generating FE meshes of the pelvis using only 10 CT slices with an overall RMS error less than 3 mm. Although we have peak errors about 12 mm in these meshes, they occur relatively far from the region of interest (the acetabulum) and will have minimal effects on the performance of the model. Considering that linear meshes usually require about 70-100 pelvic CT slices (in axial mode) to generate FE models, our method has brought a significant data reduction to the automatic mesh generation step. The method, that is fully automated except for a semi-automatic bone/tissue boundary extraction part, will bring the benefits of FE methods to the clinical environment with much reduced radiation risks and data requirement.