Clonostachys rosea 'omics profiling: identification of putative metabolite-gene associations mediating its in vitro antagonism against Fusarium graminearum.

BACKGROUND: Clonostachys rosea is an established biocontrol agent. Selected strains have either mycoparasitic activity against known pathogens (e.g. Fusarium species) and/or plant growth promoting activity on various crops. Here we report outcomes from a comparative 'omics analysis leveraging a temporal variation in the in vitro antagonistic activities of C. rosea strains ACM941 and 88-710, toward understanding the molecular mechanisms underpinning mycoparasitism. RESULTS: Transcriptomic data highlighted specialized metabolism and membrane transport related genes as being significantly upregulated in ACM941 compared to 88-710 at a time point when the ACM941 strain had higher in vitro antagonistic activity than 88-710. In addition, high molecular weight specialized metabolites were differentially secreted by ACM941, with accumulation patterns of some metabolites matching the growth inhibition differences displayed by the exometabolites of the two strains. In an attempt to identify statistically relevant relationships between upregulated genes and differentially secreted metabolites, transcript and metabolomic abundance data were associated using IntLIM (Integration through Linear Modeling). Of several testable candidate associations, a putative C. rosea epidithiodiketopiperazine (ETP) gene cluster was identified as a prime candidate based on both co-regulation analysis and transcriptomic-metabolomic data association. CONCLUSIONS: Although remaining to be validated functionally, these results suggest that a data integration approach may be useful for identification of potential biomarkers underlying functional divergence in C. rosea strains.

Draft Genome Resources for Plant-Beneficial Fungi Clonostachys rosea Strains ACM941 and 88-710.

Clonostachys rosea strains ACM941 and 88-710 are beneficial microbes recognized for their plant disease control and growth promotion properties, respectively, when applied to economically important crops. In addition to their geographical and functional overlap, the two strains also share a high degree of genetic similarity. In an effort to identify the subtleties that underlie their strain-specific applications, their genomic sequence is reported here. The genome size of ACM941 was estimated to be 56.9 Mb, encoding 17,585 putative genes, while strain 88-710 was estimated to have a 55.5 Mb genome size, containing 17,188 predicted genes. Overall, ACM941 and 88-710 share >96% of their encoded genomes, such that their strain-specific characteristics are likely encoded in either the remaining variable 4% or differentially regulated shared genes or both. These genomic sequences form a foundation for future studies aimed at identifying the genomic and metabolic machinery driving their respective beneficial properties.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.

Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum.

Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed.

Pea and lentil 7S globulin crystal structures with comparative immunoglobulin epitope mapping.

Legumes represent an affordable high protein, nutrient dense food source. However, the vast majority of legume crops contain proteins that are known allergens for susceptible individuals. These include proteins from the 7S globulin family, which comprise a vast majority of seed storage proteins. Here, the crystal structures of 7S globulins from Pisum sativum L. (pea) and Lens culinaris Medicus (lentil) are presented for the first time, including pea vicillin and convicilin, and lentil vicilin. All three structures maintain the expected 7S globulin fold, with trimeric quaternary structure and monomers comprised of ß-barrel N- and C-modules. The potential impact of sequence differences on structure and packing in the different crystal space groups is noted, with potential relevance to packing upon seed deposition. Mapping on the obtained crystal structures highlights significant Ig epitope overlap between pea, lentil, peanut and soya bean and significant coverage of the entire seed storage protein, emphasizing the challenge in addressing food allergies. How recently developed biologicals might be refined to be more effective, or how these seed storage proteins might be modified in planta to be less immuno-reactive remain challenges for the future. With legumes representing an affordable, high protein, nutrient dense food source, this work will enable important research in the context of global food security and human health on an ongoing basis.

Effect of Hospitalization on Percent Median Body Mass Index at One Year, in Underweight Youth With Restrictive Eating Disorders.

PURPOSE: Data from low-weight patients with restrictive eating disorders (EDs) treated in outpatient adolescent medicine-based ED treatment programs were analyzed to determine whether there was an association between hospitalization and gain to at least 90% median body mass index (mBMI) at 1-year follow-up. METHODS: Data were retrospectively collected for 322 low-weight (<85% mBMI at intake) patients aged 9-21 years, who presented with restrictive EDs to 14 adolescent medicine-based ED programs in 2010. Positive outcome was defined as being at least 90% mBMI (%mBMI = patient's body mass index/mBMI for age × 100) at 1-year follow-up. Association between treatment at a higher level of care and gain to at least 90% mBMI was analyzed for 140 patients who were <85% mBMI at the time of presentation, had not been previously hospitalized, and had 1-year follow-up data available. RESULTS: For patients presenting at <85% mBMI, those who were hospitalized in the year following intake had 4.0 (95% confidence interval: 1.6-10.1) times the odds of gain to at least 90% mBMI, compared with patients who were not hospitalized, when controlling for baseline %mBMI. CONCLUSION: In this national cohort of patients with restrictive EDs presenting to adolescent medicine-based ED programs at <85% mBMI, those who were hospitalized had greater odds of being at least 90% mBMI at 1-year follow-up.

Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania.

Approaches which eliminate mRNA expression directly are ideally suited for reverse genetics applications in eukaryotic microbes which are asexual diploids, such as the protozoan parasite Leishmania. RNA interference (RNAi) approaches have been successful in many species, including the related parasite Trypanosoma brucei. For RNAi tests in Leishmania, we developed improved protocols for transient and stable DNA transfection, attaining efficiencies of up to 25 and 3%, respectively. This facilitated RNAi tests at the alpha-tubulin locus, whose inhibition gives a strong lethal phenotype in trypanosomatids. However, transient or stable transfection of DNAs encoding mRNAs for an alpha-tubulin stem-loop construct and GFP to monitor transfection resulted in no effect on parasite morphology, growth or tubulin expression in Leishmania major or L. donovani. Transient transfection of a 24-nucleotide double-stranded alpha-tubulin siRNA also had no effect. Similar results were obtained in studies targeting an introduced GFP gene with a GFP stem-loop construct. These data suggest that typical RNAi strategies may not work effectively in Leishmania, and raise the possibility that Leishmania is naturally deficient for RNAi activity, like Saccharomyces cerevisae. The implications to parasite biology, gene amplification, and genetic analysis are discussed.

In vitro shuttle mutagenesis using engineered mariner transposons.

Advances in our understanding of the protozoan parasite Leishmania have been facilitated by the development of molecular and genetic tools. One powerful approach for gene identification and analysis is transposon mutagenesis. This can be performed directly in vivo, but often it is more convenient to generate transpositions in vitro for subsequent analysis in vivo, in a process termed "shuttle mutagenesis." The Drosophila element mariner is well suited for application by either route. Minimal mariner elements containing cis-acting elements required for transposition have been generated, which can be further modified to suit the needs of the experimenter. Additional genetic markers and/or reporters can be introduced, which are useful for procedures such as insertional mutagenesis, shotgun sequencing, or the generation of protein and transcriptional fusions for subsequent analysis. Active transposase can readily be generated following expression in Escherichia coli, and efficiencies of 10-3/target can be obtained, allow-ing the generation of large transposon insertion libraries suitable for subsequent screening in vivo. This chapter explains the steps necessary to purify active Mos1 transposase and conduct an in vitro transposition reaction. We also discuss some of the considerations relevant to the design and application of functional mariner elements (donor plasmids) relevant to studies in Leishmania and other organisms.

Predictors of outcome at 1 year in adolescents with DSM-5 restrictive eating disorders: report of the national eating disorders quality improvement collaborative.

PURPOSE: The National Eating Disorders Quality Improvement Collaborative evaluated data of patients with restrictive eating disorders to analyze demographics of diagnostic categories and predictors of weight restoration at 1 year. METHODS: Fourteen Adolescent Medicine eating disorder programs participated in a retrospective review of 700 adolescents aged 9-21 years with three visits, with DSM-5 categories of restrictive eating disorders including anorexia nervosa (AN), atypical AN, and avoidant/restrictive food intake disorder (ARFID). Data including demographics, weight and height at intake and follow-up, treatment before intake, and treatment during the year of follow-up were analyzed. RESULTS: At intake, 53.6% met criteria for AN, 33.9% for atypical AN, and 12.4% for ARFID. Adolescents with ARFID were more likely to be male, younger, and had a longer duration of illness before presentation. All sites had a positive change in mean percentage median body mass index (%MBMI) for their population at 1-year follow-up. Controlling for age, gender, duration of illness, diagnosis, and prior higher level of care, only %MBMI at intake was a significant predictor of weight recovery. In the model, there was a 12.7% change in %MBMI (interquartile range, 6.5-19.3). Type of treatment was not predictive, and there were no significant differences between programs in terms of weight restoration. CONCLUSIONS: The National Eating Disorders Quality Improvement Collaborative provides a description of the patient population presenting to a national cross-section of 14 Adolescent Medicine eating disorder programs and categorized by DSM-5. Treatment modalities need to be further evaluated to assess for more global aspects of recovery.

Relation among stroke knowledge, lifestyle, and stroke-related screening results.

Stroke is a leading cause of individual and public health burden in the United States. A better understanding of the relation among stroke knowledge, lifestyle, and stroke-related screening results may be useful for improving prevention efforts. This paper assesses the relation among demographics, lifestyle, stroke awareness, and the presence of stroke risk factors determined by screening tests for a select elderly population. A population of 322 participants in the 1999 World Senior Games received 1 or more free screening tests and completed a stroke awareness questionnaire. Results indicate that stroke education efforts should be targeted at the very elderly, those who have less than a college education, and those who do not have a history of chronic disease. It also may be effectively directed toward those with higher cholesterol.