Fungal Planet description sheets: 1383-1435.

Novel species of fungi described in this study include those from various countries as follows: Australia, Agaricus albofoetidus, Agaricus aureoelephanti and Agaricus parviumbrus on soil, Fusarium ramsdenii from stem cankers of Araucaria cunninghamii, Keissleriella sporoboli from stem of Sporobolus natalensis, Leptosphaerulina queenslandica and Pestalotiopsis chiaroscuro from leaves of Sporobolus natalensis, Serendipita petricolae as endophyte from roots of Eriochilus petricola, Stagonospora tauntonensis from stem of Sporobolus natalensis, Teratosphaeria carnegiei from leaves of Eucalyptus grandis × E. camaldulensis and Wongia ficherai from roots of Eragrostis curvula. Canada, Lulworthia fundyensis from intertidal wood and Newbrunswickomyces abietophilus (incl. Newbrunswickomyces gen. nov.) on buds of Abies balsamea. Czech Republic, Geosmithia funiculosa from a bark beetle gallery on Ulmus minor and Neoherpotrichiella juglandicola (incl. Neoherpotrichiella gen. nov.) from wood of Juglans regia. France, Aspergillus rouenensis and Neoacrodontium gallica (incl. Neoacrodontium gen. nov.) from bore dust of Xestobium rufovillosum feeding on Quercus wood, Endoradiciella communis (incl. Endoradiciella gen. nov.) endophytic in roots of Microthlaspi perfoliatum and Entoloma simulans on soil. India, Amanita konajensis on soil and Keithomyces indicus from soil. Israel, Microascus rothbergiorum from Stylophora pistillata. Italy, Calonarius ligusticus on soil. Netherlands, Appendopyricularia juncicola (incl. Appendopyricularia gen. nov.), Eriospora juncicola and Tetraploa juncicola on dead culms of Juncus effusus, Gonatophragmium physciae on Physcia caesia and Paracosmospora physciae (incl. Paracosmospora gen. nov.) on Physcia tenella, Myrmecridium phragmitigenum on dead culm of Phragmites australis, Neochalara lolae on stems of Pteridium aquilinum, Niesslia nieuwwulvenica on dead culm of undetermined Poaceae, Nothodevriesia narthecii (incl. Nothodevriesia gen. nov.) on dead leaves of Narthecium ossifragum and Parastenospora pini (incl. Parastenospora gen. nov.) on dead twigs of Pinus sylvestris. Norway, Verticillium bjoernoeyanum from sand grains attached to a piece of driftwood on a sandy beach. Portugal, Collybiopsis cimrmanii on the base of living Quercus ilex and amongst dead leaves of Laurus and herbs. South Africa, Paraproliferophorum hyphaenes (incl. Paraproliferophorum gen. nov.) on living leaves of Hyphaene sp. and Saccothecium widdringtoniae on twigs of Widdringtonia wallichii. Spain, Cortinarius dryosalor on soil, Cyphellophora endoradicis endophytic in roots of Microthlaspi perfoliatum, Geoglossum lauri-silvae on soil, Leptographium gemmatum from fluvial sediments, Physalacria auricularioides from a dead twig of Castanea sativa, Terfezia bertae and Tuber davidlopezii in soil. Sweden, Alpova larskersii, Inocybe alpestris and Inocybe boreogodeyi on soil. Thailand, Russula banwatchanensis, Russula purpureoviridis and Russula lilacina on soil. Ukraine, Nectriella adonidis on overwintered stems of Adonis vernalis. USA, Microcyclus jacquiniae from living leaves of Jacquinia keyensis and Penicillium neoherquei from a minute mushroom sporocarp. Morphological and culture characteristics are supported by DNA barcodes. Citation: Crous PW, Boers J, Holdom D, et al. 2022. Fungal Planet description sheets: 1383-1435. Persoonia 48: 261-371. https://doi.org/10.3767/persoonia.2022.48.08.

Characterisation of the UK high energy proton research beamline for high and ultra-high dose rate (FLASH) irradiation.

Objective. This work sets out the capabilities of the high energy proton research beamline developed in the Christie proton therapy centre for Ultra-High Dose Rate (UHDR) irradiation and FLASH experiments. It also characterises the lower limits of UHDR operation for this Pencil Beam Scanning (PBS) proton hardware.Approach. Energy dependent nozzle transmission was measured using a Faraday Cup beam collector. Spot size was measured at the reference plane using a 2D scintillation detector. Integrated depth doses (IDDs) were measured. EBT3 Gafchromic film was used to compare UHDR and conventional dose rate spots. Our beam monitor calibration methodolgy for UHDR is described. A microDiamond detector was used to determine dose rates at zref. Instantaneous depth dose rates were calculated for 70-245 MeV. PBS dose rate distributions were calculated using Folkerts and Van der Water definitions.Main results. Transmission of 7.05 ± 0.1% is achieveable corresponding to a peak instantaneous dose rate of 112.7 Gy s-1. Beam parameters are comparable in conventional and UHDR mode with a spot size ofσx= 4.6 mm,σy= 6.6 mm. Dead time in the beam monitoring electonics warrants a beam current dependent MU correction in the present configuration. Fast beam scanning of 26.4 m s-1(X) and 12.1 m s-1(Y) allows PBS dose rates of the order tens of Grays per second.Significance. UHDR delivery is possible for small field sizes and high energies enabling research into the FLASH effect with PBS protons at our facility. To our knowledge this is also the first thorough characterisation of UHDR irradiation using the hardware of this clinical accelerator at energies less than 250 MeV. The data set out in this publication can be used for designing experiments at this UK research facility and inform the possible future clinical translation of UHDR PBS proton therapy.

The sap from Euphorbia peplus is effective against human nonmelanoma skin cancers.

BACKGROUND: The sap from Euphorbia peplus, commonly known as petty spurge in the U.K. or radium weed in Australia, has been used as a traditional treatment for a number of cancers. OBJECTIVE: To determine the effectiveness of E. peplus sap in a phase I/II clinical study for the topical treatment of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and intraepidermal carcinomas (IEC). METHODS: Thirty-six patients, who had refused, failed or were unsuitable for conventional treatment, were enrolled in a phase I/II clinical study. A total of 48 skin cancer lesions were treated topically with 100-300 µL of E. peplus sap once daily for 3 days. RESULTS: The complete clinical response rates at 1 month were 82% (n = 28) for BCC, 94% (n = 16) for IEC and 75% (n = 4) for SCC. After a mean follow-up of 15 months these rates were 57%, 75% and 50%, respectively. For superficial lesions < 16 mm, the response rates after follow-up were 100% for IEC (n = 10) and 78% for BCC (n = 9). CONCLUSIONS: The clinical responses for these relatively unfavourable lesions (43% had failed previous treatments, 35% were situated in the head and neck region and 30% were > 2 cm in diameter), are comparable with existing nonsurgical treatments. An active ingredient of E. peplus sap has been identified as ingenol mebutate (PEP005). This clinical study affirms community experience with E. peplus sap, and supports further clinical development of PEP005 for the treatment of BCC, SCC and IEC.

Improvement of expression and secretion of a fungal xylanase in the rumen bacterium Butyrivibrio fibrisolvens OB156 by manipulation of promoter and signal sequences.

Promoters and signal sequences for expression and secretion of a fungal xylanase encoded by a modified Neocallimastix patriciarum xynA cDNA in the rumen bacterium, Butyrivibrio fibrisolvens OB156, were investigated. Successful expression of the fungal xylanase in OB156 was obtained using the putative xylanase promoter from B. fibrisolvens strain 49. Replacing the putative -35 region sequence (TTGCAC) of the xylanase promoter with the sequence TTGACA by mutagenesis reduced the fungal xylanase expression level 4-fold in OB156, indicating that this B. fibrisolvens strain did not efficiently recognise the E. coli consensus -35 sequence. Reduction of the spacer length between the -35 and -10 regions of the xylanase promoter from 18 to 17 base-pairs (bp) considerably increased the expression levels of the fungal enzyme in both E. coli and OB156. Insertion of a pUB110 mob promoter upstream of the xylanase promoter also significantly improved the fungal xylanase expression. Secretion of the fungal xylanase mediated by the alpha-amylase signal peptide from B. fibrisolvens strain H17c was efficient in E. coli, but very poor in OB156. An increase in the hydrophobicity of the signal sequence resulted in a 4-fold increase in the extracellular portion of the fungal xylanase in OB156, indicating marked improvement in xylanase secretion efficiency. The recombinant plasmids and xylanase expression/secretion cassettes were found to be stable in OB156 after prolonged cultivation (100 generations) in the absence of antibiotic selection. These results suggest that the rumen bacterium B. fibrisolvens can be manipulated to produce and secrete a eukaryotic extracellular protein with stable maintenance of the expression cassette in plasmid form.

Modification of a xylanase cDNA isolated from an anaerobic fungus Neocallimastix patriciarum for high-level expression in Escherichia coli.

A Neocallimastix patriciarum xylanase cDNA with the core coding sequence essentially identical to xynA was isolated and modified for high-level expression in Escherichia coli. The xylanase cDNA was truncated into individual catalytic domains, which were modified at the N-terminus. These modified xylanases were synthesised as non-fusion proteins under the control of the tac promoter. High-level expression was obtained with the modified domain II construct, accounting for approx. 25% of total cellular protein. However, with the same vector and expression cassette, expression levels of constructs containing domain I or domains I and II fused in tandem were very low. RNA analysis revealed that the striking difference in expression levels of these three constructs was not due to transcription efficiency, but was mainly related to transcript stability. Further analysis of the domain II construct revealed that the high-level expression of the domain II xylanase was largely attributed to the presence of a favourable N-terminal coding sequence, as mutation at the N-terminus of the domain II dramatically reduced the expression level. The modified domain II xylanase produced in E. coli had a specific activity of 1229 U mg-1 protein at pH 7 and 50 degrees C without purification. The availability of a recombinant fungal xylanase with high specific activity and in high yield offers a potentially attractive source of xylanase for industrial applications.

Regioselective acylation of 3-O-angeloylingenol by Candida antarctica Lipase B.

Acylation of 3-O-angeloylingenol (1) with vinyl acetate, vinyl decanoate and vinyl cinnamate, catalyzed by Candida antarctica Lipase B, was investigated. In each case, compound 1 was quantitatively and regioselectively acylated to afford a single product, 3-O-angeloyl-20-O-acetylingenol (1a), 3-O-angeloyl-20-O-decanoylingenol (1b) and 3-O-angeloyl-20-O-cinnamoylingenol (1c), respectively. The structures of the novel compounds 1b-1c were determined by MS and NMR, and product 1a by comparison of RP-HPLC and TLC with a standard. Compounds 1b-1c induced a bipolar morphology of MM96L melanoma cells at a similar concentration as compound 1, as well as having activity in inhibiting the growth of MM96L melanoma cells.

Hydroxychloroquine and chloroquine: assessing the risk of retinal toxicity.

BACKGROUND: During the past 10 years there has been an increase in the use of the antimalarial agents, chloroquine and hydroxychloroquine, in the treatment of various rheumatic diseases. METHODS: In the United States, hydroxychloroquine (Plaquenil) is one of the most commonly prescribed medications for mild to moderately severe rheumatoid arthritis. Both chloroquine and hydroxychloroquine have the potential to produce an irreversible maculopathy. However, this maculopathy occurs almost exclusively at higher than recommended doses. RESULTS: Careful dose monitoring by the rheumatologist, combined with appropriate ocular examination by the eye care professional, can significantly reduce the risk of retinopathy. CONCLUSIONS: This paper discusses the use of chloroquine and hydroxychloroquine in the treatment of rheumatoid arthritis and systemic lupus erythematosus, it summarizes their retinotoxic risk, and it outlines the diagnostic techniques used to evaluate patients treated with these medications.

Tick/host interactions for Ixodes holocyclus: role, effects, biosynthesis and nature of its toxic and allergenic oral secretions.

The Australian paralysis tick Ixodes holocyclus occurs along the eastern coast of Australia. Its interaction with a wide variety of hosts causes a serious toxicosis (tick paralysis) in domestic pets and livestock (occasionally in wildlife and humans) as well as hypersensitivity reactions in humans. Tick paralysis in animals is usually fatal in the absence of speedy antitoxin treatment and human hypersensitivity may result in life-threatening anaphylaxis. The protection of such hosts against toxic or allergic effects by vaccination or desensitisation respectively has been the objective of most of our recent research. The role, biosynthesis and nature of the paralysing toxin (holocyclotoxin) and of the allergens is gradually being elucidated. In this review, some emphasis has been placed on recent research on the interactions of humans with this tick and on the partial characterisation of the allergens.

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle.

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.

A simplified liquid chromatography and electrochemical detection method for measurement of plasma noradrenaline.

A simplified, sensitive, and reliable method for the determination of noradrenaline (NA) in plasma using high-performance liquid chromatography and electrochemical detection is described. Investigations on the stability of NA in plasma and whole blood have effected an improvement in the method for collection and extraction of NA from plasma. Recycling the mobile phase and the elimination of the addition of antioxidants during the alumina extraction has resulted in savings in the costs of reagents and assay preparation time while retaining a recovery rate of 60% and improving detector response. The sensitivity of the assay for NA was 20 pg/ml of plasma using carbon paste prepared in the laboratory.

Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli.

A cDNA expression library of the rumen fungus Neocallimastix patriciarum was made in Escherichia coli. Cellulolytic clones were identified by screening on a medium containing carboxymethylcellulose. Restriction mapping and Southern hybridization analysis of selected clones revealed three distinct cellulase cDNAs, designated celA, celB and celC. Studies on the substrate specificity showed that the enzyme encoded by celA had high activity towards amorphous and microcrystalline cellulose, while the celB and celC enzymes had relatively high activity on carboxymethylcellulose, with little activity on microcrystalline cellulose. Analysis of hydrolysis products from defined cellodextrins showed that the celB and celC enzymes hydrolysed beta-1,4-glucosidic linkages randomly, whereas the celA enzyme cleaved cellotetraose to cellobiose, and cellopentaose to cellobiose and cellotriose. Cellobiose was also the only product detectable from hydrolysis of microcrystalline cellulose by the celA enzyme. Based on substrate specificity and catalytic mode, celA appears to encode a cellobiohydrolase, while celB and celC encode endoglucanases. Northern blot hybridization analysis showed that expression of the three cellulase transcripts in N. patriciarum was induced by cellulose.

Protection of Babesia bigemina-immune animals against subsequent challenge with virulent Babesia bovis.

Two groups of cattle, one previously exposed to Babesia bigemina and one not, were challenged with Babesia bovis. The group previously infected with Babesia bigemina was only mildly affected upon challenge with B. bovis, whereas four of five of the other group were severely affected. Immunoblotting studies performed in both homologous and heterologous systems showed that there were polypeptides of similar molecular weight in both species, but species-specific polypeptides were demonstrated only in B. bovis by the homologous B. bovis reaction. B. bovis antisera reacted avidly with B. bigemina-infected erythrocytes in fluorescent-antibody assays. In contrast, B. bigemina antisera did not cross-react with B. bovis-infected erythrocytes. Two groups of splenectomized calves were immunized with an enriched antigen fraction of B. bigemina. A third group was immunized by infection with B. bigemina and treatment with a drug. One of the groups of calves immunized with the antigenic fraction of B. bigemina, the group immunized by infection with B. bigemina, and a control group were challenged with B. bovis. All control calves died, whereas 50% of the calves immunized by infection with B. bigemina and 75% of the animals immunized with the B. bigemina antigen survived. The second group immunized with the B. bigemina antigen and a control group were challenged with B. bigemina. All control animals died by day 6, whereas 50% of the vaccinates survived, the deaths occurring on days 8 and 11. The nature of the probable protective mechanism is discussed.

Temperature-regulated expression of the tac/lacl system for overproduction of a fungal xylanase in Escherichia coli.

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.

Transformation and expression of an anaerobic fungal xylanase in several strains of the rumen bacterium Butyrivibrio fibrisolvens.

AIMS: To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. METHODS AND RESULTS: Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram-positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2.46 U mg-1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0.74 U mg-1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28.7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long-term cultivation. CONCLUSIONS: A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. SIGNIFICANCE AND IMPACT OF THE STUDY: This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.