Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivo.

The feasibility of skin-friction field measurements using the global luminescent oil-film skin-friction field estimation method was evaluated for a challenging case of a supercritical airfoil model under transonic wind-tunnel conditions (freestream Mach number of 0.72) at a high Reynolds number (10 million, based on the model chord length). The oil-film thickness and skin-friction coefficient distributions were estimated over the airfoil model upper surface for a range of angles of attack (from $$-0.4^{\circ }$$ - 0 . 4 ∘ to $$2.0^{\circ }$$ 2 . 0 ∘ ), thus enabling the study of different boundary-layer stability situations with laminar–turbulent transition, including cases with shock-wave/boundary-layer interaction. Conventional pressure measurements on the surface and in the wake of the model as well as Schlieren flow visualizations were conducted to support the oil-film based investigations. In the laminar-flow regions, the oil-film thickness could be generally kept below the critical limit of roughness that would induce premature boundary-layer transition. The skin friction in this region could be estimated with a moderate confidence level, as confirmed for portions of the chord by the reasonable agreement with numerical data obtained via laminar boundary-layer computations. Moreover, the location of transition onset was evaluated from the skin-friction estimations with relatively low uncertainty, thus enabling the examination of the transition location evolution with varying angle of attack. The estimated locations of transition onset were shown to be in general agreement with reference transition locations measured via temperature-sensitive paint. On the other hand, the oil-film thickness in the turbulent-flow regions was larger than the height of the viscous sublayer, which led to an hydraulically rough surface with increased skin friction, as compared to the clean configuration. For this reason, quantitative skin-friction estimations were not feasible in the turbulent-flow regions. The global effects of the oil-film setup on the flow around the airfoil were evaluated from the estimations of the aerodynamic coefficients. In particular, it was shown that the presence of the specific base coat used for the application of the oil film already induced a significant increase in airfoil drag, as compared to the clean configuration, whereas a thin oil film led to negligible or small additional increases in drag. Based on the present observations, considerations for the further improvement of the global luminescent oil-film skin-friction field estimation method in transonic flow experiments at high Reynolds numbers are elucidated. Graphic abstract

The development of vaccines against fungi and other intracellular microbes is impeded in part by a lack of suitable adjuvants. While most current vaccines against infectious diseases preferentially induce production of antibodies, cellular immunity is essential for the resolution of fungal infections. Microbes such as fungi and Mycobacterium tuberculosis require Th17 and Th1 cells for resistance, and engage the C-type lectin receptors including Dectin-2. Herein, we discovered a novel Dectin-2 ligand, the glycoprotein Blastomyces Eng2 (Bl-Eng2). Bl-Eng2 triggers robust signaling in Dectin-2 reporter cells and induces IL-6 in human PBMC and BMDC from wild type but not Dectin-2-/- and Card9-/- mice. The addition of Bl-Eng2 to a pan-fungal subunit vaccine primed large numbers of Ag-specific Th17 and Th1 cells, augmented activation and killing of fungi by myeloid effector cells, and protected mice from lethal fungal challenge, revealing Bl-Eng2’s potency as a vaccine adjuvant. Thus, ligation of Dectin-2 by Bl-Eng-2 could be harnessed as a novel adjuvant strategy to protect against infectious diseases requiring cellular immunity.

Results from a skin-friction-field measurement method using global luminescent oil-film images, in which the oil film covers a large portion of the surface, were examined by comparing them with those obtained from conventional hot-wire measurements. The measurement processes including calibrations, oil-film image acquisition, and image processing are explained, and error sources are analyzed. The experimental results show that the skin frictions measured by both methods in a turbulent boundary layer on a flat surface agree when the oil-film thickness is less than the height of the viscous sublayer (corresponding to an oil-film thickness of less than five wall units). The uncertainty of the measurements is presented. When the measurement conditions were optimum (illumination, exposure time, frame rate, oil height, etc), the uncertainty was approximately 3%–4%.

PurposeThis study (NCT00751348) evaluated the immunogenicity and safety of a combined measles-mumps-rubella-varicella (MMRV) vaccine compared to co-administration of measles-mumps-rubella and varicella (MMR+V) vaccines in Korean children during their second year of life.Materials and MethodsHealthy children aged 11-24 months received one dose of MMRV or MMR+V. Antibody titers against measles, mumps and rubella were measured using enzyme-linked immunosorbent assay and against varicella using an immunofluorescence assay. Parents/guardians recorded adverse events in diary cards for up to 43 days post-vaccination. The primary objective was to demonstrate non-inferiority of MMRV to MMR+V for all antigens in terms of seroconversion rates (SCRs), defined as a group difference with a lower limit of the 95% confidence interval (CI)>-10%.ResultsOf 474 subjects enrolled, 458 (MMRV, 301; MMR+V, 157) were included in the according-to-protocol cohort. For measles (98.0% vs. 99.4%), rubella (99.7% vs. 100%) and varicella (98.9% vs. 100%) SCRs, the lower limits of the 95% CIs for group differences were greater than -10%; however, for mumps SCRs (88.8% vs. 94.2%), it was -10.40%. The primary objective of non-inferiority in mumps SCRs was therefore not met, although the observed group difference in a post-hoc analysis of anti-mumps antibodies using a plaque reduction neutralization assay was 0.39% with a 95% CI lower limit of -4.03%. Adverse events occurred at comparable frequencies for both groups, except for more frequent fever in MMRV recipients.ConclusionBased on the pre-specified non-inferiority criterion, SCRs of the MMRV vaccine were non-inferior to that elicited by MMR+V vaccines for all antigens except mumps.