Gliotoxin effects on fungal growth: Mechanisms and exploitation

Although initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 lg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up- and down-regulated >1.9-fold; p < 0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p < 0.05), along with concomitant resistance to diamide, were evident in A. fumigatus ∆gliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (∆sod1 and ∆yap1) were hypersensitive to exogenous gliotoxin, while ∆gsh1 was resistant. Significant gliotoxin-mediated (5 µg/ml) growth inhibition (p < 0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p < 0.001) at gliotoxin (10 lg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus DgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies

Regulation of Nonribosomal Peptide Synthesis: bis-Thiomethylation Attenuates Gliotoxin Biosynthesis in Aspergillus fumigatus

Gliotoxin is a redox-active nonribosomal peptide produced by Aspergillus fumigatus. Like many other disulfide-containing epipolythiodioxopiperazines, a bis-thiomethylated form is also produced. In the case of gliotoxin, bisdethiobis(methylthio)gliotoxin (BmGT) is formed for unknown reasons by a cryptic enzyme. Here, we identify the S-adenosylmethionine- dependent gliotoxin bis-thiomethyltransferase (GtmA), which converts dithiogliotoxin to BmGT. This activity, which is induced by exogenous gliotoxin, is only detectable in protein lysates of A. fumigatus deficient in the gliotoxin oxidoreductase, gliT. Thus, GtmA is capable of substrate bis-thiomethylation. Deletion of gtmA completely abrogates BmGT formation and we now propose that the purpose of BmGT formation is primarily to attenuate gliotoxin biosynthesis. Phylogenetic analysis reveals 124 GtmA homologs within the Ascomycota phylum. GtmA is encoded outside the gliotoxin biosynthetic cluster and primarily serves to negatively regulate gliotoxin biosynthesis. This mechanism of postbiosynthetic regulation of nonribosomal peptide synthesis appears to be quite unusual

A Pilot Study of Sidewalk Equity in Seattle Using Crowdsourced Sidewalk Assessment Data

We examine the potential of using large-scale open crowdsourced sidewalk data from Project Sidewalk to study the distribution and condition of sidewalks in Seattle, WA. While potentially noisier than professionally gathered sidewalk datasets, crowdsourced data enables large, cross-regional studies that would be otherwise expensive and difficult to manage. As an initial case study, we examine spatial patterns of sidewalk quality in Seattle and their relationship to racial diversity, income level, built density, and transit modes. We close with a reflection on our approach, key limitations, and opportunities for future work.Comment: Workshop paper presented at "The 1st ASSETS'22 Workshop on The Future or urban Accessibility (UrbanAccess'22)

Hair coat properties of donkeys, mules and horses in a temperate climate

Background There are clear differences between donkeys and horses in their evolutionary history, physiology, behaviour and husbandry needs. Donkeys are often kept in climates they are not adapted to and as such may suffer impaired welfare unless protection from the elements is provided. Objectives We provide the first direct comparison of the hair coat properties of donkeys, mules and horses living outside, throughout the year, in the temperate climate of the UK. Study Design The weight, length and width of hair were measured, across the four seasons, as indicators of the hair coat insulation properties. Methods Hair samples were taken from 42 animals: 18 donkeys (4 females, 14 males), 16 horses (6 females, 10 males), and eight mules (5 females, 3 males), in March, June, September and December. Results Donkeys’ hair coats do not significantly differ across the seasons. All three measurements of the insulation properties of the hair samples indicate that donkeys do not grow a winter coat and that their hair coat was significantly lighter, shorter and thinner than that of horses and mules in winter. In contrast the hair coats of horses changed significantly between seasons, growing thicker in winter. Main Limitations The measurements cover only a limited range of features that contribute to the thermo-regulation of an animal. Further research is needed to assess shelter preferences by behavioural measures, and absolute heat loss via thermoimaging. Conclusions Donkeys, and to a lesser extent mules, appear not to be as adapted to colder, wet climates as horses, and may therefore require additional protection from the elements, such as access to a wind and waterproof shelter, in order for their welfare needs to be met

Do You Need to Travel? Mapping Face-to-Face Communication Objectives to Technology Affordances

Computer-mediated communications (CMC) can be used as a substitute for face-to-face (FtF) meetings but their effectiveness is highly context dependent. This paper describes a theoretical framework and initial experimental design for characterizing a travel replacement threshold. This effort begins with a use case of remote engineering maintenance training, conducted in three conditions: side-by-side (physically proximate), teleconference (using off-the-shelf software), and a custom VR/AR system designed to provide the apprentice with a virtual view of both the instructor’s larger scale lab and smaller scale workbench. The research hypotheses, experimental protocol, and dependent measures are described. The task involves an instructor demonstrating a circuit board troubleshooting task to a remote apprentice. The apprentice then completes the trained task independently, and performance and subject preferences are compared across conditions. The details of this paper, the result of extensive literature review and winnowing of variables, may assist researchers exploring CMC, training, or social communication

RED experiment: an assessment of boundary layer effects in a trade winds regime on microwave and infrared propagation over the sea, The

Includes bibliographical references (pages 1364-1365).The Rough Evaporation Duct experiment aimed to see if the effects of ocean waves account for errors in modeling the ranges at which radar and infrared can detect low-flying targets

EPOCHS Paper II: The Ultraviolet Luminosity Function from 7.5<z<13.57.5<z<13.5 using 110 square arcminutes of deep, blank-field data from the PEARLS Survey and Public Science Programmes

We present an analysis of the ultraviolet luminosity function (UV LF) and star formation rate density of distant galaxies ($7.5 < z < 13.5$) in the `blank' fields of the Prime Extragalactic Areas for Reionization Science (PEARLS) survey combined with Early Release Science (ERS) data from the CEERS, GLASS and NGDEEP surveys/fields. We use a combination of SED fitting tools and quality cuts to obtain a reliable selection and characterisation of high-redshift ($z>6.5$) galaxies from a consistently processed set of deep, near-infrared imaging. Within an area of 110 arcmin$^{2}$, we identify 214 candidate galaxies at redshifts $z>6.5$ and we use this sample to study the ultraviolet luminosity function (UV LF) in four redshift bins between $7.5<z<13.5$. The measured number density of galaxies at $z=8$ and $z=9$ match those of past observations undertaken by the em Hubble Space Telescope (HST). However, towards higher redshifts we find that the evolution of the UV LF is mild, resulting in higher measured number densities of UV luminous galaxies at $z=10.5$ and $z=12.5$ compared to predictions from simulations and past HST observations. When examining the star formation rate density of galaxies at this time period, our observations are still consistent with a constant star formation efficiency, are slightly lower than previous early estimations using JWST and support galaxy driven reionization at $z\sim8$.Comment: 28 Pages, 4 Tables, 9 Figures, Submitted to Ap

Comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer (ESPAC-4): a multicentre, open-label, randomised, phase 3 trial

BACKGROUND: The ESPAC-3 trial showed that adjuvant gemcitabine is the standard of care based on similar survival to and less toxicity than adjuvant 5-fluorouracil/folinic acid in patients with resected pancreatic cancer. Other clinical trials have shown better survival and tumour response with gemcitabine and capecitabine than with gemcitabine alone in advanced or metastatic pancreatic cancer. We aimed to determine the efficacy and safety of gemcitabine and capecitabine compared with gemcitabine monotherapy for resected pancreatic cancer. METHODS: We did a phase 3, two-group, open-label, multicentre, randomised clinical trial at 92 hospitals in England, Scotland, Wales, Germany, France, and Sweden. Eligible patients were aged 18 years or older and had undergone complete macroscopic resection for ductal adenocarcinoma of the pancreas (R0 or R1 resection). We randomly assigned patients (1:1) within 12 weeks of surgery to receive six cycles of either 1000 mg/m(2) gemcitabine alone administered once a week for three of every 4 weeks (one cycle) or with 1660 mg/m(2) oral capecitabine administered for 21 days followed by 7 days' rest (one cycle). Randomisation was based on a minimisation routine, and country was used as a stratification factor. The primary endpoint was overall survival, measured as the time from randomisation until death from any cause, and assessed in the intention-to-treat population. Toxicity was analysed in all patients who received trial treatment. This trial was registered with the EudraCT, number 2007-004299-38, and ISRCTN, number ISRCTN96397434. FINDINGS: Of 732 patients enrolled, 730 were included in the final analysis. Of these, 366 were randomly assigned to receive gemcitabine and 364 to gemcitabine plus capecitabine. The Independent Data and Safety Monitoring Committee requested reporting of the results after there were 458 (95%) of a target of 480 deaths. The median overall survival for patients in the gemcitabine plus capecitabine group was 28·0 months (95% CI 23·5-31·5) compared with 25·5 months (22·7-27·9) in the gemcitabine group (hazard ratio 0·82 [95% CI 0·68-0·98], p=0·032). 608 grade 3-4 adverse events were reported by 226 of 359 patients in the gemcitabine plus capecitabine group compared with 481 grade 3-4 adverse events in 196 of 366 patients in the gemcitabine group. INTERPRETATION: The adjuvant combination of gemcitabine and capecitabine should be the new standard of care following resection for pancreatic ductal adenocarcinoma

A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism