Ergot alkaloids are specialized fungal metabolites that are important as the bases of several pharmaceuticals. Many ergot alkaloids are derivatives of lysergic acid (LA) and have vasoconstrictive activity, whereas several dihydrolysergic acid (DHLA) derivatives are vasorelaxant. The pathway to LA is established, with the P450 monooxygenase CloA playing a key role in oxidizing its substrate agroclavine to LA. We analyzed the activities of products of cloA alleles from different fungi relative to DHLA biosynthesis by expressing them in a mutant of the fungus Neosartorya fumigata that accumulates festuclavine, the precursor to DHLA. Transformants expressing CloA from Epichloë typhina ϫ Epichloë festucae, which oxidizes agroclavine to LA, failed to oxidize festuclavine to DHLA. In substrate feeding experiments, these same transformants oxidized exogenously supplied agroclavine to LA, indicating that a functional CloA was produced. A genomic clone of cloA from Claviceps africana, a sorghum ergot fungus that produces a DHLA derivative, was cloned and expressed in the festuclavine-accumulating mutant of N. fumigata, but several introns in this genomic clone were not processed properly. Expression of a synthetic intron-free version of C. africana cloA resulted in the accumulation of DHLA as assessed by fluorescence high-pressure liquid chromatography (HPLC) and liquid chromatographymass spectrometry (LC-MS). In substrate feeding experiments, the C. africana CloA also accepted agroclavine as the substrate, oxidizing it to LA. The data indicate that a specialized allele of cloA is required for DHLA biosynthesis and that the pharmaceutically important compound DHLA can be produced in engineered N. fumigata. IMPORTANCE Ergot alkaloids are fungal metabolites that have impacted humankind historically as poisons and more recently as pharmaceuticals used to treat dementia, migraines, and other disorders. Much is known about the biosynthesis of ergot alkaloids that are derived from lysergic acid (LA), but important questions remain about a parallel pathway to ergot alkaloids derived from dihydrolysergic acid (DHLA). DHLA-derived alkaloids have minor structural differences compared to LA-derived alkaloids but can have very different activities. To understand how DHLA is made, we analyzed activities of a key enzyme in the DHLA pathway and found that it differed from its counterpart in the LA pathway. Our data indicate a critical difference between the two pathways and provide a strategy for producing DHLA by modifying a model fungus. The ability to produce DHLA in a model fungus may facilitate synthesis of DHLA-derived pharmaceuticals.KEYWORDS ergot alkaloids, lysergic acid, dihydrolysergic acid, P450 monooxygenase E rgot alkaloids are specialized fungal metabolites that have impacted humankind historically as toxins and more recently as pharmaceuticals used to treat dementia, migraines, hyperprolactinemia, and Parkinson's disease (1-4). Many natural ergot alka-

Neosartorya fumigata (Aspergillus fumigatus) is the most common cause of invasive aspergillosis, a frequently fatal lung disease primarily affecting immunocompromised individuals. This opportunistic fungal pathogen produces several classes of specialised metabolites including products of a branch of the ergot alkaloid pathway called fumigaclavines. The biosynthesis of the N. fumigata ergot alkaloids and their relation to those produced by alternate pathway branches in fungi from the plant-inhabiting Clavicipitaceae have been well-characterised, but the potential role of these alkaloids in animal pathogenesis has not been studied extensively. We investigated the contribution of ergot alkaloids to virulence of N. fumigata by measuring mortality in the model insect Galleria mellonella. Larvae were injected with conidia (asexual spores) of two different wild-type strains of N. fumigata and three different ergot alkaloid mutants derived by previous gene knockouts and differing in ergot alkaloid profiles. Elimination of all ergot alkaloids significantly reduced virulence of N. fumigata in G. mellonella (P < 0.0001). Mutants accumulating intermediates but not the pathway end product fumigaclavine C also were less virulent than the wild type (P < 0.0003). The data indicate that ergot alkaloids contribute to virulence of N. fumigata in this insect model and that fumigaclavine C is important for full virulence.

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Purpose: To evaluate the ability of the PDI Check (PDI Check LLC, Anchorage, AK) near vision screening game to assess monocular acuity, stereopsis, suppression, and color. Methods: Children and adults consented to perform the PDI Check Quick Screening game following conventional near testing of patched Rosenbaum acuity, Titmus Fly stereo, Worth 4-dot, and Ishihara color. Time to complete each test and preferred method were recorded. Results: A total of 77 patients (5 to 63 years old) attempted all tests. There was a positive correlation between the PDI Check and conventional tests for all visual tasks. Using previously determined instrument referral criteria, sensitivity/specificity was determined for right acuity (67%/91%), left acuity (55%/94%), stereopsis (87%/95%), red-green color (80%/99%), and ocular suppression (58%/98%). Screening time was 202 ± 96 versus 99 ± 42 seconds for the PDI Check and the game was preferred by 87%. Conclusions: The PDI Check provided a valid assessment of near vision in less than half the time of conventional testing without patches or goggles. This Quick Screening version may help eye technicians and physicians with time efficiency in the frequent task of near visual assessment. [ J Pediatr Ophthalmol Strabismus . 2019;56(4):234–237.]

ObjectiveAccurate estimation of hyperopia and astigmatism is challenging in delayed children. Conventional skiascopy holds rows of increasing power ± lenses vertically in front of one eye. The school bus accommodation-relaxing skiascopy (SBA-RS) design holds child-friendly, lenses +1 to +10D horizontally so that a higher power fogs the nontested eye-relaxing accommodation without cycloplegia.MethodsDesign: Evaluation of diagnostic test. Subjects: Patients undergoing comprehensive eye examination in a pediatric ophthalmology practice. Cycloplegic (cyclopentolate 1%) retinoscopy was compared to dry SBA-RS and Retinomax (Righton, Japan) during pediatric eye examinations. Outcome measures: correlations, Chi-square and receiver operating characteristic (ROC) curve.ResultsOf 470 patients with a median age 6 years, 238 were under the age of 60 months and 110 had developmental delays. For those with cycloplegic spherical equivalent hyperopia over 0.7 D, median (90% CI) value for retinoscopy was +2.63 D (+0.75, +6.88), for SBA-RS was +2.50 D (+0.50, +6.75) and less for 184 with Retinomax +1.88 D (−1.56, +6.13) but similar despite delays. Astigmatic cylinder SBA-RS +1.50 D (+0.25, +4.00) lagged retinoscopy +1.75 D (+0.75,+4.50) but Retinomax was greater +2.00 D (+0.25, +4.64). Cycloplegic refractive components such as spherical equivalent, cylinder, and J0 and J45 power vectors correlated highly and were near unity with SBA-RS and Retinomax with the latter deviating greater. SBA-RS screened for amblyopia risk factors up to 92% sensitive and 94% specific.ConclusionAccommodation-relaxing horizontal skiascopy very precisely estimates astigmatism power and axis and only lags cycloplegic refraction by about 0.15D in hyperopic patients fairly independent of neurodevelopmental delay. This technique can quickly estimate refraction even in delayed patients potentially reducing some need for cycloplegia.Clinical Trials RegistryNCT03668067.

Background: Handheld devices can automatically give an estimate of refraction. The established method for refraction comparison using spherical equivalent (M) and J0, J45 vector transformations by Bland-Altman analysis is too complex for non-eye doctors involved with vision screening and remote vision clinics. Therefore, a simpler comparison technique was developed. Methods: Based on the spectacle limit to resolve grade A 1 logMAR, B 3 logMAR and C 6 logMAR blur, J0, J45, and M are combined into the Alaska Blind Child Discovery (ABCD) composite ellipsoid GRADE system. Pediatric eye patients had confirmatory examination after dry refraction with three portable autorefractors: Plusoptix, 2WIN and Retinomax. The refractions were then compared using both Bland-Altman and ABCD composite. Performance to detect AAPOS amblyopia risk factors was also assessed. Results: A total of 202 children, mean age seven years, 28% high spectacle need and 43% AAPOS 2013 amblyopia risk factors showed high correlation with cycloplegic refraction (intraclass correlation 0.49 to 0.90) for sphere, J0 and J45 spectacle components. Plusoptix had more (10%) inconclusives due to patients out-of-range. The Retinomax was unable to screen some younger children and was less reliable for sphere but gave more precise astigmatism estimates. The proportion of autorefractions expected to give GRADE A/B highneed patients acuity improvement to 20/40 would be 41% for Plusoptix, 39% for 2WIN and 65% for Retinomax. Sensitivity/specificity for amblyopia risk factor detection was 80%/83% for Plusoptix, 72%/88% for 2WIN and 84%/73% for Retinomax. Conclusion:The simplified spectacle comparison resembled Bland-Altman and could assist lay vision screeners and non-eye doctors attempting remote spectacle donation worldwide.

Families of infants born with congenital defects such as cleft lip, cleft palate, and combinations of these often grasp with accepting the condition. Nurses can help facilitate acceptance by parents and families and play a key role in the management and habilitation of these children.

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