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The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces , based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium -mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R . graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.

Background/aimsThere is a paucity of high-level evidence to support the management of childhood uveitis, particularly for those children without juvenile idiopathic arthritis uveitis (JIA). We undertook a modified Delphi consensus exercise to identify agreement in the management of chronic anterior uveitis (CAU), the most common manifestation of childhood disease.MethodsA four-round, two-panel process was undertaken between June and December 2017. Paediatric uveitis specialists identified through multiple sources, including a multicentre network (the Paediatric Ocular Inflammation Group), were invited to participate. They were asked whether they agreed with items derived from existing guidelines on the management of JIA-U when extrapolated to the population of all children with CAU. Consensus was defined as agreement greater than or equal to 75% of respondents.Results26 of the 38 (68%) invited specialists participated with the exercise, and response rates were 100% for rounds one to three, and 92% for round four. Consensus was reached on 23 of the 44 items. Items for which consensus was not reached included management at presentation, use of systemic and periocular steroids for children with severe disease and the role of conventional steroid sparing immunosuppressants beyond methotrexate.ConclusionThe areas of management uncertainty at the level of the group, as indicated by absence of consensus, reflect the areas where the evidence base is particularly poor. Our findings identify the key areas for the future research needed to ensure better outcomes for this blinding childhood ocular inflammatory disorders.

Cisplatin is an effective chemotherapeutic drug for the treatment of bladder cancer, though cisplatin-induced nephrotoxicity (CIN) occurs in approximately 20-30% of patients, limiting its clinical use. Evidence has shown that cytochrome P450 2E1 (CYP2E1), a drug metabolism enzyme expressed in proximal tubules, mediates the production of reactive oxygen species (ROS) during cisplatin-induced injury. Previously, we showed that the repurposed drug 4-methylpyrazole (4MP; fomepizole) blocks CYP2E1 activity and prevents acetaminophen-induced liver injury. Here, we investigated the potential protective effects of 4MP against CIN. Male and female C57BL/6J mice were treated with a single 20 mg/kg dose of cisplatin for 3 days (acute) or 9 mg/kg/week for 4 weeks (repeated dosing regimen) with or without a co-treatment of 50 mg/kg 4MP. Our findings revealed that acute treatment with cisplatin induced severe histological tubular damage and elevated plasma BUN and creatinine levels in male mice, but not in female mice. This difference correlated with higher basal CYP2E1 expression in the kidneys of male mice compared to female mice. We also found that cisplatin increased renal CYP2E1 activity and that inhibition of CYP2E1 with 4MP significantly reduced cisplatin induced cell death in male mice and primary normal human kidney cells. By contrast, human bladder cancer cells do not express CYP2E1, and treatment with 4MP did not interfere with cisplatin anti-cancer effects in human bladder cancer HTB9 cells. This study highlights the critical role of CYP2E1 in CIN and suggests that its inhibition with 4MP in the kidney is a potential prophylactic therapeutic option to prevent CIN in bladder cancer patients without affecting its anti-neoplastic effect.