Next Generation Sequencing Workshop at the Royal Society of Medicine (London, May 2022): how genomics is on the path to modernizing genetic toxicology.

The use of error-corrected Next Generation Sequencing (ecNG) to determine mutagenicity has been a subject of growing interest and potentially a disruptive technology that could supplement, and in time, replace current testing paradigms in preclinical safety assessment. Considering this, a Next Generation Sequencing Workshop was held at the Royal Society of Medicine in London in May 2022, supported by the United Kingdom Environmental Mutagen Society (UKEMS) and TwinStrand Biosciences (WA, USA), to discuss progress and future applications of this technology. In this meeting report, the invited speakers provide an overview of the Workshop topics covered and identify future directions for research. In the area of somatic mutagenesis, several speakers reviewed recent progress made with correlating ecNGS to classic in vivo transgenic rodent mutation assays as well as exploring the use of this technology directly in humans and animals, and in complex organoid models. Additionally, ecNGS has been used for detecting off-target effects of gene editing tools and emerging data suggest ecNGS potential to measure clonal expansion of cells carrying mutations in cancer driver genes as an early marker of carcinogenic potential and for direct human biomonitoring. As such, the workshop demonstrated the importance of raising awareness and support for advancing the science of ecNGS for mutagenesis, gene editing, and carcinogenesis research. Furthermore, the potential of this new technology to contribute to advances in drug and product development and improve safety assessment was extensively explored.

Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity of DYRK1B mutants from cancer and metabolic syndrome.

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B(S421A) mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B(D239A) mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS(G12V). In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.

A novel DYRK1B inhibitor AZ191 demonstrates that DYRK1B acts independently of GSK3ß to phosphorylate cyclin D1 at Thr(286), not Thr(288).

DYRK1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B) is amplified in certain cancers and may be an oncogene; however, our knowledge of DYRK1B has been limited by the lack of selective inhibitors. In the present study we describe AZ191, a potent small molecule inhibitor that selectively inhibits DYRK1B in vitro and in cells. CCND1 (cyclin D1), a key regulator of the mammalian G1-S-phase transition, is phosphorylated on Thr(286) by GSK3ß (glycogen synthase kinase 3ß) to promote its degradation. DYRK1B has also been proposed to promote CCND1 turnover, but was reported to phosphorylate Thr(288) rather than Thr(286). Using in vitro kinase assays, phospho-specific immunoblot analysis and MS in conjunction with AZ191 we now show that DYRK1B phosphorylates CCND1 at Thr(286), not Thr(288), in vitro and in cells. In HEK (human embryonic kidney)-293 and PANC-1 cells (which exhibit DYRK1B amplification) DYRK1B drives Thr(286) phosphorylation and proteasome-dependent turnover of CCND1 and this is abolished by AZ191 or DYRK1B RNAi, but not by GSK3ß inhibitors or GSK3ß RNAi. DYRK1B expression causes a G1-phase cell-cycle arrest, but overexpression of CCND1 (wild-type or T286A) fails to overcome this; indeed, DYRK1B also promotes the expression of p21CIP1 (21 kDa CDK-interacting protein 1) and p27KIP1 (CDK-inhibitory protein 1). The results of the present study demonstrate for the first time that DYRK1B is a novel Thr(286)-CCND1 kinase that acts independently of GSK3ß to promote CCND1 degradation. Furthermore, we anticipate that AZ191 may prove useful in defining further substrates and biological functions of DYRK1B.

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3).

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC6(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.

Uptake of the OMERACT-OARSI Hip and Knee Osteoarthritis Core Outcome Set: Review of Randomized Controlled Trials from 1997 to 2017.

OBJECTIVE: To assess the uptake of the OMERACT-OARSI (Outcome Measures in Rheumatology- Osteoarthritis Research Society International) core outcome set (COS) domains in hip and/or knee osteoarthritis (OA) trials. METHODS: There were 382 trials of hip and/or knee OA identified from the ClinicalTrial.gov registry from 1997 to 2017. Frequency of COS adoption was assessed by year and per 5-yearly phases. RESULTS: COS adoption decreased from 61% between 1997 and 2001 to 38% between 2012 and 2016. Pain (95%) and physical function (86%) were most consistently adopted. Patient's global assessment (48%) was the principal missing domain. CONCLUSION: Limited adoption of the COS domains indicates that further consideration to improve uptake is required.

The OMERACT-OARSI Core Domain Set for Measurement in Clinical Trials of Hip and/or Knee Osteoarthritis.

OBJECTIVE: To update the 1997 OMERACT-OARSI (Outcome Measures in Rheumatology-Osteoarthritis Research Society International) core domain set for clinical trials in hip and/or knee osteoarthritis (OA). METHODS: An initial review of the COMET database of core outcome sets (COS) was undertaken to identify all domains reported in previous COS including individuals with hip and/or knee OA. These were presented during 5 patient and health professionals/researcher meetings in 3 continents (Europe, Australasia, North America). A 3-round international Delphi survey was then undertaken among patients, healthcare professionals, researchers, and industry representatives to gain consensus on key domains to be included in a core domain set for hip and/or knee OA. Findings were presented and discussed in small groups at OMERACT 2018, where consensus was obtained in the final plenary. RESULTS: Four previous COS were identified. Using these, and the patient and health professionals/researcher meetings, 50 potential domains formed the Delphi survey. There were 426 individuals from 25 different countries who contributed to the Delphi exercise. OMERACT 2018 delegates (n = 129) voted on candidate domains. Six domains gained agreement as mandatory to be measured and reported in all hip and/or knee OA clinical trials: pain, physical function, quality of life, and patient's global assessment of the target joint, in addition to the mandated core domain of adverse events including mortality. Joint structure was agreed as mandatory in specific circumstances, i.e., depending on the intervention. CONCLUSION: The updated core domain set for hip and/or knee OA has been agreed upon. Work will commence to determine which outcome measurement instrument should be recommended to cover each core domain.

DYRK1A-mediated Cyclin D1 Degradation in Neural Stem Cells Contributes to the Neurogenic Cortical Defects in Down Syndrome.

Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insights into the mechanisms that couple cell cycle regulation and neuron production in cortical neural stem cells, emphasizing that the deleterious effect of DYRK1A triplication in the formation of the cerebral cortex begins at the onset of neurogenesis, which is relevant to the search for early therapeutic interventions in Down syndrome.

Apoplastic permeability of sclerotia of Sclerotium rolfsii, Sclerotium cepivorum and Rhizoctonia solani.

Intact mature sclerotia of Sclerotium rolfsii sacc, and Sclerotium cepivorum Berk, produced in culture are impermeable to the apoplastic tracer sulphorhodamine G. Both of these species produce sclerotia with rinds. Some movement of sulphorhodamine into sclerotia of Rhizoctonia solani Kühn, which have no rind, occurred but the fluorochrome was arrested after permeation of at most the outer five layers of cells. In all cases, low permeability depended on an intact outer layer, and when sclerotia of each species were bisected to provide direct access of sulphorhodamine to all tissue layers, the fluorochrome permeated the cell walls and extracellular matrix (where present) of many cells within the sclerotium. A marked reduction in permeability of intact sclerotia occurs at maturity in a number of species and might he important in long-term Survival.

Biology of mycorrhizal associations of epacrids (Ericaceae).

Epacrids, a group of southern hemisphere plants formerly considered members of the separate family Epacridaceae, are in fact most closely allied to the Vaccinioid tribe (Ericaceae). Epacrids and other extant ericoid mycorrhiza-forming plants appear to have a monophyletic origin. In common with many Ericaceae they form ericoid mycorrhizas. ITS sequence data indicate that the fungi forming ericoid mycorrhizas with epacrids and other extant Ericaceae are broadly similar, belonging to a poorly defined group of ascomycetes with phylogenetic affinities to Helotiales. The basic development and structure of ericoid mycorrhizal infections in epacrids is similar to other Ericaceae. However, data are limited on the structure and physiology of both hair roots and ericoid mycorrhizas for all Ericaceae. Relatively little is known about the functional significance of ericoid mycorrhizas in epacrids in southern hemisphere habitats that are often poor in organic matter accumulation. However the abilities of fungal endophytes of epacrids to utilize organic N and P substrates equal those of endophytes from northern hemisphere heathland plant hosts. Investigations using 15 N/13 C-labelled organic N substrates suggest that mycorrhizal endophytes are important, at least, to the N nutrition of their epacrid hosts in some habitats. Contents Summary 305 I. Epacrid plant hosts 306 II. Evolution of ericoid mycorrhizas in epacrids 306 III. Epacrid hair roots and their mycorrhizal associations 307 IV. Seasonality and incidence of mycorrhizal infection 310 V. Structure and development of mycorrhizal associations 311 VI. Nature of the mycorrhizal fungal endophytes 315 VII. Community and population biology of mycorrhizal endophytes 318 VIII. Functional aspects of mycorrhizas in epacrids 319 IX. Conclusions 322 Acknowledgements 322 References 322.

Armillaria luteobubalina mycelium develops air pores that conduct oxygen to rhizomorph clusters.

Armillaria luteobubalina produces air pores in culture. They consist of two parts: a basal region of tissue elevated to form a mound covered with a rind continuous with that of the colony, but perforated; and an apical region of long parallel hyphae, cemented together by scattered patches of extracellular material. This forms a hydrophobic structure that is elevated above the general level of the mycelial crust and does not easily become waterlogged. Air pores develop near the inoculum plug shortly after inoculation, arising directly from the mycelium, and rhizomorphs are initiated from them. The air pore contains a complex system of gas space connecting the atmosphere with the central canal of each rhizomorph. The tissue beneath the melanised colony crust also contains gas space, especially near air pores. This is also connected with the gas space of each rhizomorph and of each air pore. Measurements with oxygen electrodes show that air pores and their associated rhizomorphs conduct oxygen. The average oxygen conductance of a group of air pores with associated rhizomorphs, within agar blocks, but with rhizomorph apices cut off, was about 700 x 10(-12) m3s(-1), equivalent to about 200 x 10(-12) m3s(-1) for each air-pore. We conclude that the air pores conduct oxygen into the gas space below the pigmented mycelium of the colony, where the rhizomorphs - which also conduct oxygen - originate. A. luteobubalina thus has a complex aerating system which allows efficient diffusion of oxygen into rhizomorphs, and this is likely to facilitate extension of inoculum into low-oxygen environments.

Changes in vacuolar and mitochondrial motility and tubularity in response to zinc in a Paxillus involutus isolate from a zinc-rich soil.

Short-term effects of zinc on organelles were investigated in Paxillus involutus from a zinc-rich soil. Vacuoles were labelled with Oregon Green 488 carboxylic acid and mitochondria with DiOC(6)(3). Hyphae were treated with ZnSO(4) in the range 1-100 mM and examined by fluorescence microscopy. ZnSO(4) caused loss of tubularity and motility in both organelles depending on concentration and exposure time. Tubular vacuoles thickened after 15 min in 5 mM ZnSO(4) and became spherical at higher concentrations. Mitochondria fragmented after 30 min in 25 mM ZnSO(4). Vacuoles recovered their tubularity after transfer to reverse osmosis water depending on ZnSO(4) concentration and exposure time during treatment. Mitochondria recovered their tubularity with time, both with and without removal of the ZnSO(4) solution. K(2)SO(4) (as control) had no effect on vacuoles but disrupted mitochondria, the effect also depending on concentration and duration of exposure.

Regulators of GTP-binding proteins cause morphological changes in the vacuole system of the filamentous fungus, Pisolithus tinctorius.

Tubule formation is a widespread feature of the endomembrane system of eukaryotic cells, serving as an alternative to the better-known transport process of vesicular shuttling. In filamentous fungi, tubule formation by vacuoles is particularly pronounced, but little is known of its regulation. Using the hyphae of the basidiomycete Pisolithus tinctorius as our test system, we have investigated the effects of four drugs whose modulation, in animal cells, of the tubule/vesicle equilibrium is believed to be due to the altered activity of a GTP-binding protein (GTP gamma S, GDP beta S, aluminium fluoride, and Brefeldin A). In Pisolithus tinctorius, GTP gamma S, a non-hydrolysable form of GTP, strongly promoted vacuolar tubule formation in the tip cell and next four cells. The effects of GTP gamma S could be antagonised by pre-treatment of hyphae with GDP beta S, a non-phosphorylatable form of GDP. These results support the idea that a GTP-binding protein plays a regulatory role in vacuolar tubule formation. This could be a dynamin-like GTP-ase, since GTP gamma S-stimulated tubule formation has only been reported previously in cases where a dynamin is involved. Treatment with aluminium fluoride stimulated vacuolar tubule formation at a distance from the tip cell, but NaF controls indicated that this was not a GTP-binding-protein specific effect. Brefeldin A antagonised GTP gamma S, and inhibited tubule formation in the tip cell. Given that Brefeldin A also affects the ER and Golgi bodies of Pisolithus tinctorius, as shown previously, it is not clear yet whether the effects of Brefeldin A on the vacuole system are direct or indirect.

Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures.

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mM inorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [(13)C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and (13)C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched (13)C in extraradical hyphae was recovered in the fatty acid 16:1omega5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system in G. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.