Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus Verticillium dahliae by Agrobacterium tumefaciens-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacteriumtumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses

A systematic review on integration mechanisms in human and animal health surveillance systems with a view to addressing global health security threats

Lymphatic filariasis and onchocerciasis are neglected tropical diseases (NTDs) targeted for elimination by mass (antifilarial) drug administration. These drugs are predominantly active against the microfilarial progeny of adult worms. New drugs or combinations are needed to improve patient therapy and to enhance the effectiveness of interventions in persistent hotspots of transmission. Several therapies and regimens are currently in (pre-)clinical testing. Clinical trial simulators (CTSs) project patient outcomes to inform the design of clinical trials but have not been widely applied to NTDs, where their resource-saving payoffs could be highly beneficial. We demonstrate the utility of CTSs using our individual-based onchocerciasis transmission model (EPIONCHO-IBM) that projects trial outcomes of a hypothetical macrofilaricidal drug. We identify key design decisions that influence the power of clinical trials, including participant eligibility criteria and post-treatment follow-up times for measuring infection indicators. We discuss how CTSs help to inform target product profiles

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762. Findings Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months. Interpretation Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function. Funding UK Stroke Association and NIHR Health Technology Assessment Programme