Cleaning the Cellular Factory:Deletion of McrA in Aspergillus oryzae NSAR1 and the generation of a novel kojic acid deficient strain for cleaner heterologous production of secondary metabolites

The use of filamentous fungi as cellular factories, where natural product pathways can be refactored and expressed in a host strain, continues to aid the field of natural product discovery. Much work has been done to develop host strains which are genetically tractable, and for which there are multiple selectable markers and controllable expression systems. To fully exploit these strains, it is beneficial to understand their natural metabolic capabilities, as such knowledge can rule out host metabolites from analysis of transgenic lines and highlight any potential interplay between endogenous and exogenous pathways. Additionally, once identified, the deletion of secondary metabolite pathways from host strains can simplify the detection and purification of heterologous compounds. To this end, secondary metabolite production in Aspergillus oryzae strain NSAR1 has been investigated via the deletion of the newly discovered negative regulator of secondary metabolism, mcrA (multicluster regulator A). In all ascomycetes previously studied mcrA deletion led to an increase in secondary metabolite production. Surprisingly, the only detectable phenotypic change in NSAR1 was a doubling in the yields of kojic acid, with no novel secondary metabolites produced. This supports the previous claim that secondary metabolite production has been repressed in A. oryzae and demonstrates that such repression is not McrA-mediated. Strain NSAR1 was then modified by employing CRISPR-Cas9 technology to disrupt the production of kojic acid, generating the novel strain NSARΔK, which combines the various beneficial traits of NSAR1 with a uniquely clean secondary metabolite background

Prevalence of Trachoma in Gambella Region, Ethiopia: Results of Three Population-Based Prevalence Surveys Conducted with the Global Trachoma Mapping Project.

PURPOSE: In Ethiopia, trachoma is a major public health problem, accounting for 11.5% of all cases of blindness. In Gambella, one of the country's most remote regions, the 2005-2006 National Survey of Blindness, Low Vision and Trachoma estimated a region-level prevalence of active trachoma of 19.1% in those aged 1-9 years. Detailed district or sub-regional level estimates are required to implement interventions. METHODS: Population-based prevalence surveys were carried out following a 2-stage cluster random sampling methodology and Global Trachoma Mapping Project protocols. As the 13 districts (woredas) in Gambella had relatively small populations, they were grouped together to form three evaluation units (EUs) of about 100,000 persons each, and all subsequent survey planning and sampling was carried out at EU-level. RESULTS: Altogether, 558 cases of TF (17.2%) were identified in 3238 children aged 1-9 years across the three EUs. The adjusted TF prevalences in 1-9-year-olds for the three EUs were 11.5%, 12.5% and 19.3%; 14.4% for Gambella overall. A total of 142 cases of trichiasis (3.8%) were identified among 3781 adults aged 15 years or older, with age- and sex-adjusted EU-level trichiasis prevalences in adults being 0.8%, 1.3% and 2.4%; 1.5% overall. CONCLUSION: The high prevalences of TF and trichiasis throughout Gambella indicate a need for rapid scaling up of the World Health Organization SAFE strategy (surgery, antibiotics, facial cleanliness, and environmental improvement) to help meet the 2020 target of global elimination of trachoma as a public health problem

Prevalence of Trachoma in Benishangul Gumuz Region, Ethiopia: Results of Seven Population-Based Surveys from the Global Trachoma Mapping Project.

PURPOSE: Trachoma is a major cause of blindness in Ethiopia, and targeted for elimination as a public health problem by the year 2020. Prevalence data are needed to plan interventions. We set out to estimate the prevalence of trachoma in each evaluation unit of grouped districts ("woredas") in Benishangul Gumuz region, Ethiopia. METHODS: We conducted seven cross-sectional community-based surveys, covering 20 woredas, between December 2013 and January 2014, as part of the Global Trachoma Mapping Project (GTMP). The standardized GTMP training package and methodologies were used. RESULTS: A total of 5828 households and 21,919 individuals were enumerated in the surveys. 19,583 people (89.3%) were present when survey teams visited. A total of 19,530 (99.7%) consented to examination, 11,063 (56.6%) of whom were female. The region-wide age- and sex-adjusted trichiasis prevalence in adults aged ≥15 years was 1.3%. Two evaluation units covering four woredas (Pawe, Mandura, Bulen and Dibate) with a combined rural population of 166,959 require implementation of the A, F and E components of the SAFE strategy (surgery, antibiotics, facial cleanliness and environmental improvement) for at least three years before re-survey, and intervention planning should begin for these woredas as soon as possible. CONCLUSION: Both active trachoma and trichiasis are public health problems in Benishangul Gumuz, which needs implementation of the full SAFE strategy

Estimating the Intracluster Correlation Coefficient for the Clinical Sign "Trachomatous Inflammation-Follicular" in Population-Based Trachoma Prevalence Surveys: Results From a Meta-Regression Analysis of 261 Standardized Preintervention Surveys Carried Out in Ethiopia, Mozambique, and Nigeria.

Sample sizes in cluster surveys must be greater than those in surveys using simple random sampling in order to obtain similarly precise prevalence estimates, because results from subjects examined in the same cluster cannot be assumed to be independent. Therefore, a crucial aspect of cluster sampling is estimation of the intracluster correlation coefficient (ρ): the degree of relatedness of outcomes in a given cluster, defined as the proportion of total variance accounted for by between-cluster variation. In infectious disease epidemiology, this coefficient is related to transmission patterns and the natural history of infection; its value also depends on particulars of survey design. Estimation of ρ is often difficult due to the lack of comparable survey data with which to calculate summary estimates. Here we use a parametric bootstrap model to estimate ρ for the ocular clinical sign "trachomatous inflammation-follicular" (TF) among children aged 1-9 years within population-based trachoma prevalence surveys. We present results from a meta-regression analysis of data from 261 such surveys completed using standardized methods in Ethiopia, Mozambique, and Nigeria in 2012-2015. Consistent with the underlying theory, we found that ρ increased with increasing overall TF prevalence and smaller numbers of children examined per cluster. Estimates of ρ for TF were independently higher in Ethiopia than in the other countries

Low Prevalence of Ocular Chlamydia trachomatis Infection and Active Trachoma in the Western Division of Fiji.

BACKGROUND: Trachoma is the leading infectious cause of blindness and is caused by ocular infection with the bacterium Chlamydia trachomatis (Ct). While the majority of the global disease burden is found in sub-Saharan Africa, the Western Pacific Region has been identified as trachoma endemic. Population surveys carried out throughout Fiji have shown an abundance of both clinically active trachoma and trachomatous trichiasis in all divisions. This finding is at odds with the clinical experience of local healthcare workers who do not consider trachoma to be highly prevalent. We aimed to determine whether conjunctival infection with Ct could be detected in one administrative division of Fiji. METHODS: A population-based survey of 2306 individuals was conducted using the Global Trachoma Mapping Project methodology. Population prevalence of active trachoma in children and trichiasis in adults was estimated using the World Health Organization simplified grading system. Conjunctival swabs were collected from 1009 children aged 1-9 years. DNA from swabs was tested for the presence of the Ct plasmid and human endogenous control. RESULTS: The prevalence of active trachoma in 1-9 year olds was 3.4%. The age-adjusted prevalence was 2.8% (95% CI: 1.4-4.3%). The unadjusted prevalence of ocular Ct infection in 1-9 year-olds was 1.9% (19/1009), and the age-adjusted infection prevalence was 2.3% (95% CI: 0.4-2.5%). The median DNA load was 41 Ct plasmid copies per swab (min 20, first quartile 32, mean 6665, third quartile 161, max 86354). There was no association between current infection and follicular trachoma. No cases of trachomatous trichiasis were identified. DISCUSSION: The Western Division of Fiji has a low prevalence of clinical trachoma. Ocular Ct infections were observed, but they were predominantly low load infections and were not correlated with clinical signs. Our study data suggest that trachoma does not meet the WHO definition of a public health problem in this Division of Fiji, but the inconsistency with previous studies warrants further investigation

A multicenter study on chronic cough in children: Burden and etiologies based on a standardized management pathway

Background: While the burden of chronic cough in children has been documented, etiologic factors across multiple settings and age have not been described. In children with chronic cough, we aimed (1) to evaluate the burden and etiologies using a standar

Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi

Has charge symmetry breaking been observed in the d d -> alpha pi0 reaction?

Estimates are made of the d d -> alpha gamma gamma production cross sections in a model where each neutron-proton pair in the beam and target initiates an n p -> d gamma reaction. This approach, which successfully reproduces observables in two-pion production at intermediate energies, suggests that direct two-photon production could provide a very significant background to the measurement of the charge-symmetry-breaking (CSB) reaction d d -> alpha pi0. A non-vanishing CSB cross section has been reported which might be confused with such two-photon production under the given experimental conditions.Comment: 9 pages, ReVTEX, 2 figures, submitted to Phys. Rev. Let

Prevalence of and Risk Factors for Trachoma in Southern Nations, Nationalities, and Peoples' Region, Ethiopia: Results of 40 Population-Based Prevalence Surveys Carried Out with the Global Trachoma Mapping Project.

PURPOSE: We sought to estimate the prevalence of trachoma at sufficiently fine resolution to allow elimination interventions to begin, where required, in the Southern Nations, Nationalities, and Peoples' Region (SNNPR) of Ethiopia. METHODS: We carried out cross-sectional population-based surveys in 14 rural zones. A 2-stage cluster randomized sampling technique was used. A total of 40 evaluation units (EUs) covering 110 districts ("woredas") were surveyed from February 2013 to May 2014 as part of the Global Trachoma Mapping Project (GTMP), using the standardized GTMP training package and methodology. RESULTS: A total of 30,187 households were visited in 1047 kebeles (clusters). A total of 131,926 people were enumerated, with 121,397 (92.0%) consenting to examination. Of these, 65,903 (54.3%) were female. In 38 EUs (108 woredas), TF prevalence was above the 10% threshold at which the World Health Organization recommends mass drug administration with azithromycin annually for at least 3 years. The region-level age- and sex-adjusted trichiasis prevalence was 1.5%, with the highest prevalence of 6.1% found in Cheha woreda in Gurage zone. The region-level age-adjusted TF prevalence was 25.9%. The highest TF prevalence found was 48.5% in Amaro and Burji woredas. In children aged 1-9 years, TF was associated with being a younger child, living at an altitude 15°C, and the use of open defecation by household members. CONCLUSION: Active trachoma and trichiasis are significant public health problems in SNNPR, requiring full implementation of the SAFE strategy (surgery, antibiotics, facial cleanliness, and environmental improvement)

Prevalence of Trachoma in Kano State, Nigeria: Results of 44 Local Government Area-Level Surveys.

PURPOSE: We sought to determine the prevalence of trachoma in 44 Local Government Areas (LGAs) of Kano State, Nigeria. METHODS: A population-based prevalence survey was conducted in each Kano LGA. We used a two-stage systematic and quasi-random sampling strategy to select 25 households from each of 25 clusters in each LGA. All consenting household residents aged 1 year and above were examined for trachomatous inflammation-follicular (TF), trachomatous inflammation-intense (TI) and trichiasis. RESULTS: State-wide crude prevalence of TF in persons aged 1-9 years was 3.4% (95% CI 3.3-3.5%), and of trichiasis in those aged ≥15 years was 2.3% (95% CI 2.1-2.4%). LGA-level age- and sex-adjusted trichiasis prevalence in those aged ≥15 years ranged from 0.1% to 2.9%. All but 4 (9%) of 44 LGAs had trichiasis prevalences in adults above the elimination threshold of 0.2%. State-wide prevalence of trichiasis in adult women was significantly higher than in adult men (2.6% vs 1.8%; OR = 1.5, 95% CI 1.3-1.7; p = 0.001). Four of 44 LGAs had TF prevalences in 1-9-year-olds between 10 and 15%, while another six LGAs had TF prevalences between 5 and 9.9%. In 37 LGAs, >80% of households had access to water within 30 minutes round-trip, but household latrine access was >80% in only 19 LGAs. CONCLUSION: Trichiasis is a public health problem in most LGAs in Kano. Surgeons need to be trained and deployed to provide community-based trichiasis surgery, with emphasis on delivery of such services to women. Antibiotics, facial cleanliness and environmental improvement are needed in 10 LGAs