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Genome sequencing of filamentous fungi has demonstrated that most secondary metabolite biosynthetic gene clusters (BGCs) are silent under standard laboratory conditions. In this work, we have established an in vitro CRISPR-Cas9 system in

ObjectiveNumerous factors are considered to impact on the rate of complications during salvage total laryngectomy procedures. Neck dissection could be one of these factors. This study analysed the pattern of lymph node metastasis and rate of occult neck disease during salvage total laryngectomy as well as the impact of neck dissection on survival and complication rates.MethodThis was a retrospective analysis of a prospectively maintained laryngectomy database in two large tertiary teaching hospitals.ResultsThe rate of occult neck disease was 11.1 per cent. Most cases with occult neck disease had rT4 disease. Patients with complications, advanced tumour stage and positive margins had a significant decrease in overall survival. Patients receiving elective neck dissection did not have any survival benefit. Positron emission tomography-computed tomography showed a very high specificity and negative predictive value.ConclusionAccording to the low risk of occult neck disease when using contemporary imaging techniques as well as the lack of impact on survival, conservative management of the neck should be considered for crT1-T3 recurrence.

Functions of protein SUMOylation remain incompletely understood in different cell types. The budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, but dynein-pathway components were not identified as SUMO-targets in the filamentous fungusAspergillus nidulans. ViaA. nidulansforward genetics, here we identifiedubaBQ247*, a loss-of-function mutation in a SUMO-activation enzyme UbaB. Colonies of theubaBQ247*, ΔubaBand ΔsumOmutants looked similar and less healthy than the wild-type colony. In these mutants, about 10% of nuclei are connected by abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. Nuclei connected by chromatin bridges are mostly in interphase, suggesting that these bridges do not prevent cell-cycle progression. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO-targets being nuclear proteins, for example, topoisomerase II whose SUMOylation defect gives rise to chromatin bridges in mammalian cells. Unlike in mammalian cells, however, loss of SUMOylation inA. nidulansdoes not apparently affect the metaphase-to-anaphase transition, further highlighting differences in the requirements of SUMOylation in different cell types. Finally, loss of UbaB or SumO does not affect dynein-and LIS1-mediated early-endosome transport, indicating that SUMOylation is unnecessary for dynein or LIS1 function inA. nidulans.

For many years, two central, unanswered questions in cytoskeleton research were how microtubule assembly is nucleated and microtubule polarity established. The discoveries of γ-tubulin and γ-tubulin ring complexes were key advances that allowed these questions to be substantially answered. The discovery of γ-tubulin was the product of a genetic screen in Aspergillus nidulans for genes important for microtubule function. γ-Tubulin is a member of the tubulin superfamily of proteins, closely related to α- and β-tubulin but distinct from both. It is ubiquitous in eukaryotes, and in many organisms there are small families of γ-tubulin genes. γ-Tubulin and associated proteins form ring-like complexes that localize to microtubule-organizing centers (MTOCs) and play an important role in the nucleation of microtubule assembly from MTOCs and the establishment of microtubule polarity.

Waste plastics represent major environmental and economic burdens due to their ubiquity, slow breakdown rates, and inadequacy of current recycling routes. Polyethylenes are particularly problematic, because they lack robust recycling approaches despite being the most abundant plastics in use today. We report a novel chemical and biological approach for the rapid conversion of polyethylenes into structurally complex and pharmacologically active compounds. We present conditions for aerobic, catalytic digestion of polyethylenes collected from post-consumer and oceanic waste streams, creating carboxylic diacids that can then be used as a carbon source by the fungus Aspergillus nidulans. As a proof of principle, we have engineered strains of A. nidulans to synthesize the fungal secondary metabolites asperbenzaldehyde, citreoviridin, and mutilin when grown on these digestion products. This hybrid approach considerably expands the range of products to which polyethylenes can be upcycled.

The microtubule-associated protein tau promotes the stabilization of the axonal cytoskeleton in neurons. In several neurodegenerative diseases, such as Alzheimer's disease, tau has been found to dissociate from microtubules, leading to the formation of pathological aggregates that display an amyloid fibril-like structure. Recent structural studies have shown that the tau filaments isolated from different neurodegenerative disorders have structurally distinct fibril cores that are specific to the disease. These "strains" of tau fibrils appear to propagate between neurons in a prion-like fashion that maintains their initial template structure. In addition, the strains isolated from diseased tissue appear to have structures that are different from those made by the most commonly used in vitro modeling inducer molecule, heparin. The structural differences among strains in different diseases and in vitro-induced tau fibrils may contribute to recent failures in clinical trials of compounds designed to target tau pathology. This study identifies an isoquinoline compound (ANTC-15) isolated from the fungus Aspergillus nidulans that can both inhibit filaments induced by arachidonic acid (ARA) and disassemble preformed ARA fibrils. When compared to a tau aggregation inhibitor currently in clinical trials (LMTX, LMTM, or TRx0237), ANTC-15 and LMTX were found to have opposing inducer-specific activities against ARA and heparin in vitro-induced tau filaments. These findings may help explain the disappointing results in translating potent preclinical inhibitor candidates to successful clinical treatments.

Introduction Vancomycin treats serious Gram-positive infections such as methicillin-resistant Staphylococcus aureus. In St George’s University Hospital’s (SGH) intensive care unit (ICU) settings, vancomycin is administered by continuous infusion. Steady-state serum concentrations are measured daily with a 20-25 mg/L target. Non-therapeutic concentrations are associated with adverse drug reactions/prolonged length of stay.1 A SGH service-evaluation conducted across all three ICUs, revealed variable adherence to/effectiveness of its vancomycin prescribing/administration/monitoring protocol.2 Consequently, multifaceted interventions were devised using the Institute-for-Healthcare-Improvement’s model Plan-Do-Study-Act (PDSA) cycles and piloted on General ICU (GICU). Aim (1) To improve adherence to/effectiveness of the vancomycin protocol. (2) To ascertain administration accuracy of paper-fluid-balance-charts compared to the electronic-prescribing-and-medicines-administration (ePMA) system to assist with identifying per protocol treated patients. Methods PDSA Cycle-1 was conducted over a 9-month period (09/2021-05/2021) in which a mix of system/person-focused interventions were implemented. Protocol dosing2 was revised, introducing a >90kg patient 2g loading dose, renal-function category revision and increased maintenance dose for creatinine clearance (CLCR) >90ml/min. Protocol accessibility was increased via integration into an ePMA prescribing interface, plus CliniBee/Microguide apps. Educational slides on relevant protocol aspects were incorporated into medical/nursing induction training. Data relating to vancomycin prescribing/administration/monitoring for all non-renal replacement patients was extracted retrospectively from the ePMA system between 09/2021-05/2022. This data was compared to baseline GICU data (07/2020-07/2021).2 The project and associated interventions were approved by Trust Clinical Governance and Audit Teams. Data was collected by pharmacists directly involved in patient’s care and stored/analysed on the Trust’s secure server in line with Data Protection Act principles. Due to local generalisability, ethics approval wasn’t required. Results Compared to baseline, the proportion of patients receiving per protocol prescribing/administration of loading/maintenance doses with daily monitoring, nearly doubled (39% (7/18) to 68% (15/22)). 48-hour vancomycin serum concentrations in all patients increased therapeutically by 21% (3/9 to 7/13). In per protocol treated patients, concentrations increased 15% (2/7 to 4/9) therapeutically, decreased 21% (3/7 to 2/9) supra-therapeutically and increased 4% (2/7 to 3/9) sub-therapeutically. Supra-therapeutic concentrations were associated with CLCR90ml/min and obesity. Compared to the ePMA system, there was 36% (8/22) less paper-fluid-balance-charts recording both loading/maintenance doses. Maintenance dose administration times in 9% (2/22) of cases differed by >60 minutes. Discussion/Conclusion Staff-turnover periods were associated with decreased protocol compliance. Observations suggest further education is required around prescribing/administration of standardised infusion bags for maintenance dosing. Pharmacist integration into daily Microbiology ward rounds may increase protocol compliance. Higher 20mg/kg loading doses for obese patients and maintenance dose revision should be considered to reduce non-therapeutic concentrations.3 Limitations include heterogenous/small sample sizes due to data paucity and vancomycin requirement. This can be addressed by involving all ICU’s in PDSA Cycle-2. Due to disparities, both the ePMA system and paper-fluid-balance-charts should be used to identify protocol adherence. Utilisation of digital-infusion-pump data to quantify administration accuracy may offer a promising solution. Piloted multifaceted interventions were successful at improving adherence to/effectiveness of the vancomycin protocol. Findings have informed further interventions and data capture methods for PDSA Cycle-2 implementation across all ICUs. References 1. Perin N, Roger C, Marin G, Molinari N, Evrard A, Lavigne J et al. Vancomycin Serum Concentration after 48 h of Administration: A 3-Years Survey in an Intensive Care Unit. Antibiotics. 2020;9(11):793. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698174/ 2. Oakley R, Bakrania P, Yau T, Standing J, Lonsdale D. P37 Variable adherence to and effectiveness of a vancomycin continuous infusion protocol within ICUs at a London tertiary-care hospital: a single-centre retrospective service evaluation. JAC-Antimicrobial Resistance. 2022;4(Supplement_1). Available from: https://academic.oup.com/jacamr/issue/4/Supplement_1 3. Rybak M, Le J, Lodise T, Levine D, Bradley J, Liu C et al. Therapeutic monitoring of vancomycin for serious methicillin-resistant Staphylococcus aureus infections: A revised consensus guideline and review by the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, the Pediatric Infectious Diseases Society, and the Society of Infectious Diseases Pharmacists. American Journal of Health-System Pharmacy. 2020;77(11):835-864. Available from: https://academic.oup.com/ajhp/article/77/11/835/5810200?login=false

Every year in the training seminar on working psychodynamically and developmentally with Haverford College students, where I (Philip) work, we read Henry Seiden’s (2009) now classic paper “On the L...

On September 9th, 2019 we lost our dear friend and colleague, Greg Eells, to suicide. The one-year marker of his death passed at a juncture in time when the world felt like it was upside down. It s...

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA (mcrAΔ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG, a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrAΔ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrAΔ generally resulted in greater upregulation of SMs than upregulation of llmG, indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG. However, the combination of mcrAΔ and upregulation of llmG generally resulted in greater compound production than mcrAΔ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.

SUMOylation, covalent attachment of the small ubiquitin-like modifier protein SUMO to proteins, regulates protein interactions and activity and plays a crucial role in the regulation of many key cellular processes. Understanding the roles of SUMO in these processes ultimately requires identification of the proteins that are SUMOylated in the organism under study. The filamentous fungus Aspergillus nidulans serves as an excellent model for many aspects of fungal biology, and it would be of great value to determine the proteins that are SUMOylated in this organism (i.e. its SUMOylome). We have developed a new and effective approach for identifying SUMOylated proteins in this organism in which we lock proteins in their SUMOylated state, affinity purify SUMOylated proteins using the high affinity S-tag, and identify them using sensitive Orbitrap mass spectroscopy. This approach allows us to distinguish proteins that are SUMOylated from proteins that are binding partners of SUMOylated proteins or are bound non-covalently to SUMO. This approach has allowed us to identify 149 proteins that are SUMOylated in A. nidulans. Of these, 67 are predicted to be involved in transcription and particularly in the regulation of transcription, 21 are predicted to be involved in RNA processing and 16 are predicted to function in DNA replication or repair.

Established in 1950, the Association for University and College Counseling Center Directors (AUCCCD) is an international organization comprising universities and colleges (https://www.auccc...

Giardia duodenalis is one of the most common food and water-borne intestinal parasites of humans and animals worldwide. Fresh, ready-to-eat produce such as leafy greens and salad mixes are considered potential transmission vehicles for Giardia infection in humans. Therefore, a specific, sensitive, and reliable method for Giardia detection in leafy greens is needed. We optimized washing procedures for the recovery of Giardia cysts from leafy greens and adapted and validated an existing EF1α LAMP assay for the detection of Giardia DNA to support routine diagnostic surveillance and disease outbreak investigations. Four leafy green types (35 ± 1 g) were spiked with 100 Giardia cysts and we compared washing by shaking with 1 M glycine (n = 20) or 0.1% Alconox (n = 20). DNA was extracted from washes, tested by LAMP and melt curve analysis, and time to positive (TTP) values compared. The detection limit was determined by spiking 10 (n = 40) Giardia cysts onto these same types of leafy greens and processing as above with 0.1% Alconox. Method robustness was assessed by subjecting spring mix (n = 45 total) to aging (1, 3 or 7 days) and washes to aging and freezing conditions prior to testing. Assay repeatability and specificity were evaluated, and an artificial positive control (APC) distinguishable by melt temperature (Tm) from DNA of Giardia spiked on leafy greens was designed to rule out cross-contamination from the control. Giardia detection rates were higher and TTP was lower (P, Highlights • We adapted and validated EF1α LAMP assay for detection of Giardia in leafy greens. • Giardia LAMP is sensitive, specific and has a faster turnaround time than nPCR. • The assay performs well on aged and deteriorated produce samples. • Giardia LAMP has been implemented in our lab for routine surveillance testing.

Objective The global coronavirus disease 2019 (COVID-19) pandemic has necessitated rapid alterations to diagnostic pathways for head and neck cancer patients that aim to reduce risk to patients (exposure to the hospital environment) and staff (aerosol-generating procedures). Transoral fine needle aspiration cytology offers a low-risk means of rapidly diagnosing patients with oral cavity or oropharyngeal lesions. The technique was utilised in selected patients at our institution during the pandemic. The outcomes are considered in this study. Method Diagnostic outcomes were retrospectively evaluated for a series of patients undergoing transoral fine needle aspiration cytology of oral cavity and oropharyngeal lesions during the COVID-19 pandemic. Results Five patients underwent transoral fine needle aspiration cytology, yielding lesional material in 100 per cent, with cell blocks providing additional information. In one case, excision biopsy of a lymphoproliferative lesion was required for final diagnosis. Conclusion Transoral fine needle aspiration cytology can provide rapid diagnosis in patients with oral cavity and oropharyngeal lesions. Whilst limitations exist (including tolerability and lesion location), the technique offers significant advantages pertinent to the COVID-19 era, and could be employed in the future to obviate diagnostic surgery in selected patients.

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC, and P21WAF-1, key regulators of Notch/Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1&ndash, mRNA interactions, which could block the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans. One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

This narrative review highlights the therapeutic significance of topical corticosteroid (TCS) vehicles and provides subsequent guidance to improve clinical and research outcomes. A greater understanding of the relationship between the topical vehicle, corticosteroid and skin is needed to ensure safer, more effective treatment for patients. Topical vehicles are not inert and can affect TCS bioavailability, due to the ability of their composition to positively or negatively influence skin status and change the physiochemical characteristics of an inherent corticosteroid. However, this principle is not commonly understood, and has contributed to inconsistencies in potency classification systems. This review provides an insight into the research methods and standardization needed to determine TCS product bioavailability. It identifies formulation components responsible for vehicle composition that underpin the quality, stability, compounding and functionalities of vehicle ingredients. This helps to contextualize how topical vehicles can be responsible for clinically significant effects, and how their composition gives products unique properties. In turn, this facilitates a more in-depth understanding of which resources offer information to inform the best selection of TCS products and why products should be prescribed by brand or manufacturer. This review will better equip clinicians and formulary teams to appraise products. It will also inform prescribing of Specials and why products should not be manipulated. The recommendations, accompanied by patient perspectives on using TCS products, assist clinical decision-making. They also identify the need for research into concomitant application of TCS products with other topical therapies.

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen A. fumigatus. By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in A. nidulans. This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in Aspergillus species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism.

Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus. Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits., Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulans sensu stricto clinical isolates. Just as in Aspergillus fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity. IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus. Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC, and P21WAF-1, key regulators of Notch/Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1–mRNA interactions, which could block the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans. One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

Supplementary Figure S1. The clinical isolates MO80069 and SP-2605-48 are metabolically different from the FGSC-A4 reference strain. Principal component analysis (PCA) of the levels of quantified metabolites identified in biological replicates of all three strains when grown for 16 h in minimal medium supplemented with glucose (A), ethanol (B), acetate (C) and mucin (D).Supplementary Figure S2. The clinical isolates MO80069 and SP-2605-48 are metabolically different from the FGSC-A4 reference strain. Hierarchical component analysis (HCA) of the levels of quantified metabolites identified in biological replicates of all three strains when grown for 16 h in minimal medium supplemented with glucose (A), ethanol (B), acetate (C) and mucin (D). Supplementary Figure S3. The A. nidulans clinical isolates are sensitive to hydrogen peroxide-induced oxidative stress but not to other physiological-relevant stresses. Strains were grown in tenfold serial dilutions in complete medium supplemented with increasing concentrations of H2O2 for 48 h at 37°C (A), or they were grown from 105spores on glucose minimal medium (GMM) supplemented with increasing concentrations of menadione (B), in the presence of iron-depleted GMM supplemented with the iron chelators BPS and ferrozine (C) and on GMM in the presence of different temperatures (D) for 5 days at 37°C. Standard deviations represent biological triplicates with *pSupplementary Figure S4. The clinical isolates MO80069 and SP-2605-48 are true A. nidulans strains. The phylogenetic tree was constructed based on the CaM, BenA, RPB2 and ITS rDNA sequencing data, which was compared to sequences from other species in the Aspergillus section of Nidulantes.Supplementary Table S1. Identified metabolites in the A. nidulansclinical isolates MO80069 and SP-2605-48 and the reference strain FGSC-A4 when grown for 24 h in minimal medium (MM) supplemented with fructose and after transfer to MM supplemented with either glucose, acetate, ethanol or mucin for 16 h. Also shown are the average and standard deviations for normalised (divided by fungal dryweight) metabolite quantities measured in 4 biological replicates and corresponding p-values (p-value < 0.05 was considered significant in ) when comparing metabolite quantity in each clinical isolate to the quantity in the reference strain. In addition fold change of metabolite quantity between the clinical isolates and the reference strain was calculated for all significantly different metabolite quantities. Supplementary Table S2. Single nucleotide polymorphisms (SNPs) detected in the genomes of the A. nidulans clinical isolates MO80069 and SP-2605-48 in comparison to the reference strain FGSC A4 and to each other. Nucleotide and amino acid details of each SNP are shown as well as the impact of the resulting SNP mutation. Supplementary Table S3. Large scale insertions and deletions (indels) detected in the genomes of the A. nidulans clinical isolates MO80069LAB and SP-2605-48. Genomes were compared to the reference genome of strain FGSC A4. Gene IDs are given if the indels span predicted open reading frames (ORFs). Also shown are the chromosomal location of the indel, the DNA sequence in the reference strain, the DNA sequence of the indel ("=" signifies that the sequence in the clinical isolate is the same as in the reference strain) and the number of basepairs for each indel ("imprecise" denotes all sequences containing an "N", which is a base that could not be identified during sequencing). Individual tabs show indels that are specific to either strain or that are present in both strains. Supplementary Table S4. Number and type of mutation in genes encoding enzymes involved in carbohydrate (C) metabolism, sexual reproduction and cell wall biosynthesis and degradation. Also indicated is the clinical isolate in which the mutation occurs and the resulting nucleotide and amino acid changes.

Background Food insecurity (FI) impacted 15.7% (5.9 million) of US households with children in 2017. These households often experience issues within one or more of the food security pillars: access, availability, utilisation or stability. An underexplored area within the pillar of utilisation that may impact FI risk is the availability of kitchen equipment in households. This exploratory project aimed to quantify household food preparation equipment ownership and use by household FI status. Methods An online platform (Qualtrics, Provo, UT, USA) was used to administer a questionnaire to a sample of 135 parents of children aged 11–14 years. The instrument queried sociodemographic characteristics, food preparation items owned and frequency of use of 44 items within a 6‐month interval. Household FI was measured using the 18‐item US Department of Agriculture, Household Food Security Survey Module with a 12‐month reference period. Results Households experiencing FI (n = 39; 28.9%) owned an average of five fewer items than their food secure counterparts (n = 96, 71.1%, P 0.01). Conclusions Compared to food secure households, the number of food preparation items owned was lower, although frequency of use was the same, in US households that were FI. Future projects should investigate how food equipment ownership impacts cooking behaviours, and whether households experiencing FI display behaviours to compensate for a differing set of equipment.

We have identified the cyclin domain-containing proteins encoded by the genomes of 17 species ofAspergillusas well as 15 members of other genera of filamentous ascomycetes. Phylogenetic analyses reveal that the cyclins fall into three groups, as in other eukaryotic phyla, and, more significantly, that they are remarkably conserved in these fungi. All 32 species examined, for example, have three group I cyclins, cyclins that are particularly important because they regulate the cell cycle, and these are highly conserved. Within the group I cyclins there are three distinct clades, and each fungus has a single member of each clade. These findings are in marked contrast to the yeastsSaccharomyces cerevisiae, Schizosaccharomyces pombe, andCandida albicans, which have more numerous group I cyclins. These results indicate that findings on cyclin function made with a modelAspergillusspecies, such asA. nidulans, are likely to apply to other Aspergilli and be informative for a broad range of filamentous ascomycetes. In this regard, we note that the functions of only oneAspergillusgroup I cyclin have been analysed (NimECyclin BofA. nidulans). We have consequently carried out an analysis of the members of the other two clades usingA. nidulansas our model. We have found that one of these cyclins, PucA, is essential, but deletion of PucA in a strain carrying a deletion of CdhA, an activator of the anaphase promoting complex/cyclosome (APC/C), is not lethal. These data, coupled with data from heterokaryon rescue experiments, indicate that PucA is an essential G1/S cyclin that is required for the inactivation of the APC/C-CdhA, which, in turn, allows the initiation of the S phase of the cell cycle. Our data also reveal that PucA has additional, non-essential, roles in the cell cycle in interphase. TheA. nidulansmember of the third clade (AN2137) has not previously been named or analyzed. We designate this geneclbA. ClbA localizes to kinetochores from mid G2 until just prior to chromosomal condensation. Deletion ofclbAdoes not affect viability. However, by using a regulatable promoter system new toAspergillus, we have found that expression of a version of ClbA in which the destruction box sequences have been removed is lethal and causes a mitotic arrest and a high frequency of non-disjunction. Thus, although ClbA is not essential, its timely destruction is essential for viability, chromosomal disjunction, and successful completion of mitosis.

The soybean cyst nematode (SCN)–soybean pathosystem is often reproduced under greenhouse conditions for basic research purposes, increasing inoculum, and soybean germplasm screening. Application of pesticides for SCN bioassays is often necessary for keeping insect and mite pest populations under control. The research presented here looks at the application of several foliar pesticides (active ingredients; abamectin, bifenthrin, clofentezine, imidacloprid, kinoprene, and pyridaben), a soil-applied imidacloprid, and combinations of pesticides on bioassays to determine if pesticide applications have a negative impact on SCN female and cyst numbers as well as egg and second-stage juvenile (J2) production, which could lead to dubious results. Results from this experiment demonstrate these chemicals can be used to control pest populations on soybean without significantly altering SCN female, cyst, egg, and J2 counts.

Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici ΔPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development. This article is protected by copyright. All rights reserved.

Several commercially available alternative sweeteners have potential in reducing the caloric content of baked products. Sugar alcohols and natural sweeteners have similar bulk as sucrose and can replace sucrose directly. High intensity sweeteners have high potency but light weight so bulking agents are often added. This study determined alternative sweeteners and combinations of alternative sweetener and a bulking agent that produced good quality white layer cakes.; Results: Cakes made with maltitol were acceptable but erythritol and fructose produced undesirable cakes. Maltodextrin and polydextrose were acceptable bulking agents, producing cakes that were similar to control cakes. The flavor of cakes sweetened with sucralose was acceptable but those with stevia had a disagreeable metallic aftertaste. Cakes made with sucralose plus maltodextrin were preferred over those containing sucralose plus polydextrose. Consumer acceptance of flavor, texture and overall liking of cakes containing maltitol was similar to sucrose and both were preferred over cakes containing maltodextrin plus sucralose.; Conclusion: Replacing sucrose with maltitol in white layer cakes reduced the caloric content by 16% with no loss in quality. © 2016 Society of Chemical Industry.; © 2016 Society of Chemical Industry.

Summary Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation.

For eukaryotes like fungi to regulate biological responses to environmental stimuli, various signalling cascades are utilized, like the highly conserved mitogen‐activated protein kinase (MAPK) pathways. In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module regulates development and the production of secondary metabolites (SMs). This pathway consists five proteins, the three kinases SteC, MkkB and MpkB, the adaptor SteD and the scaffold HamE. In this study, homologs of these five pheromone module proteins have been identified in the plant and human pathogenic fungus Aspergillus flavus. We have shown that a tetrameric complex consisting of the three kinases and the SteD adaptor is assembled in this species. It was observed that this complex assembles in the cytoplasm and that MpkB translocates into the nucleus. Deletion of steC, mkkB, mpkB or steD results in abolishment of both asexual sporulation and sclerotia production. This complex is required for the positive regulation of aflatoxin production and negative regulation of various SMs, including leporin B and cyclopiazonic acid (CPA), likely via MpkB interactions in the nucleus. These data highlight the conservation of the pheromone module in Aspergillus species, signifying the importance of this pathway in regulating fungal development and secondary metabolism.