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Background: Wastewater-based epidemiology (WBE) is rapidly developing as a powerful public health tool. It can provide information about a wide range of health determinants (HDs), including community exposure to environmental hazards, trends in consumption of licit and illicit substances, spread of infectious diseases, and general community health. As such, the list of possible candidate HDs for WBE is almost limitless. Consequently, a means to evaluate and prioritize suitable candidates for WBE is useful, particularly for public health authorities, who often face resource constraints., Objectives: We have developed a framework to assist public health authorities to decide what HDs may be appropriate for WBE and what biomarkers could be used. This commentary reflects the experience of the authors, who work at the interface of research and public health implementation., Discussion: To be suitable for WBE, a candidate HD should address a public health or scientific issue that would benefit from better understanding at the population level. For HDs where information on individual exposures or stratification by population subgroups is required, WBE is less suitable. Where other methodologies are already used to monitor the candidate HD, consideration must be given to whether WBE could provide better or complementary information to the current approach. An essential requirement of WBE is a biomarker specific for the candidate HD. A biomarker in this context refers to any human-excreted chemical or biological that could act as an indicator of consumption or exposure to an environmental hazard or of the human health state. Suitable biomarkers should meet several criteria outlined in this commentary, which requires background knowledge for both the biomarker and the HD. An evaluation tree summarizing key considerations for public health authorities when assessing the suitability of candidate HDs for WBE and an example evaluation are presented. https://doi.org/10.1289/EHP11115.

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signaling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localize the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P 2 , PI[4]P and PI[4,5]P 2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homolog of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did enable the detection of PI[3,4,5]P 3 in septa and mycelium of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P 3 in ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P 3 and that fungal PTEN homologs are functional lipid phosphatases. The F. oxysporum chemotropism defect suggests a conserved role of PTEN homologs in chemotaxis across protists, fungi and mammals., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Epichloë festucae is an endophytic fungus that forms a symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from uninfected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem MS was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using MarVis in combination with the BioCyc and KEGG databases, and an in-house Epichloë metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative MS analysis of apoplastic wash fluid from wild-type- vs mutant-infected plants identified a novel Leu/Ile glycoside metabolite present in the former., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi., (© 2020 John Wiley & Sons Ltd.)

Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium-like structure known as an expressorium. In plant-pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild-type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium-like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring-like structure at the point of cuticle exit as found in the wild-type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Epichloë festucae forms mutualistic symbiotic interactions with grasses of the Lolium and Festuca genera. Protection from insect and mammalian herbivory are the best-documented host benefits of these associations. The two main classes of anti-mammalian alkaloids synthesized by E. festucae are the ergot alkaloids and indole diterpenes, of which ergovaline and lolitrems are the principal terminal products. Synthesis of both metabolites require multiple gene products encoded by clusters of 11 genes located at the subtelomeric regions of chromosomes I and III respectively. These loci are essentially unexpressed in axenic culture but among the most highly expressed genes in planta. We show here that heterochromatin 1 protein (HepA) is an important component of the regulatory machinery that maintains these loci in a silent state in culture. Deletion of this gene led to derepression of eas and ltm gene expression under non-symbiotic culture conditions. Although there was no obvious culture phenotype, RNAseq analysis revealed that around 1000 genes were differentially expressed in the ΔhepA mutant compared to wild type with just one-third upregulated. Inoculation of the ΔhepA mutants into seedlings of Lolium perenne led to a severe host interaction phenotype characterized by a reduction in tiller length but an increase in tiller number. Hyphae within the leaves of these associations were much more abundant in the intercellular spaces of the leaves and aberrantly colonized the vascular bundles. This physiological change was accompanied by a dramatic change in the transcriptome with around 900 genes differentially expressed, with two thirds of these upregulated. This major physiological change was accompanied by a decrease in ltm gene expression and loss of the ability to synthesize lolitrems. These results show that HepA has an important role in controlling the chromatin state of these sub-telomeric secondary metabolite genes, including their symbiosis-specific regulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Epichloë festucae is an endophyte of the agriculturally important perennial ryegrass. This species systemically colonises the aerial tissues of this host where its growth is tightly regulated thereby maintaining a mutualistic symbiotic interaction. Recent studies have suggested that small secreted proteins, termed effectors, play a vital role in the suppression of host defence responses. To date only a few effectors with important roles in mutualistic interactions have been described. Here we make use of the fully assembled E. festucae genome and EffectorP to generate a suite of 141 effector candidates. These were analysed with respect to their genome location and expression profiles in planta and in several symbiosis-defective mutants. We found an association between effector candidates and a class of transposable elements known as MITEs, but no correlation with other dynamic features of the E. festucae genome, such as transposable element-rich regions. Three effector candidates and a small GPI-anchored protein were chosen for functional analysis based on their high expression in planta compared to in culture and their differential regulation in symbiosis defective E. festucae mutants. All three candidate effector proteins were shown to possess a functional signal peptide and two could be detected in the extracellular medium by western blotting. Localization of the effector candidates in planta suggests that they are not translocated into the plant cell, but rather, are localized in the apoplastic space or are attached to the cell wall. Deletion and overexpression of the effector candidates, as well as the putative GPI-anchored protein, did not affect the plant growth phenotype or restrict growth of E. festucae mutants in planta. These results indicate that these proteins are either not required for the interaction at the observed life stages or that there is redundancy between effectors expressed by E. festucae., Competing Interests: The authors have declared that no competing interests exist.

Epichloë grass endophytes comprise a group of filamentous fungi of both sexual and asexual species. Known for the beneficial characteristics they endow upon their grass hosts, the identification of these endophyte species has been of great interest agronomically and scientifically. The use of simple sequence repeat loci and the variation in repeat elements has been used to rapidly identify endophyte species and strains, however, little is known of how the structure of repeat elements changes between species and strains, and where these repeat elements are located in the fungal genome. We report on an in-depth analysis of the structure and genomic location of the simple sequence repeat locus B10, commonly used for Epichloë endophyte species identification. The B10 repeat was found to be located within an exon of a putative bZIP transcription factor, suggesting possible impacts on polypeptide sequence and thus protein function. Analysis of this repeat in the asexual endophyte hybrid Epichloë uncinata revealed that the structure of B10 alleles reflects the ancestral species that hybridized to give rise to this species. Understanding the structure and sequence of these simple sequence repeats provides a useful set of tools for readily distinguishing strains and for gaining insights into the ancestral species that have undergone hybridization events.

Beneficial associations between plants and microbes play an important role in both natural and agricultural ecosystems. For example, associations between fungi of the genus Epichloë, and cool-season grasses are known for their ability to increase resistance to insect pests, fungal pathogens and drought. However, little is known about the molecular changes induced by endophyte infection. To study the impact of endophyte infection, we compared the expression profiles, based on RNA sequencing, of perennial ryegrass infected with Epichloë festucae with noninfected plants. We show that infection causes dramatic changes in the expression of over one third of host genes. This is in stark contrast to mycorrhizal associations, where substantially fewer changes in host gene expression are observed, and is more similar to pathogenic interactions. We reveal that endophyte infection triggers reprogramming of host metabolism, favouring secondary metabolism at a cost to primary metabolism. Infection also induces changes in host development, particularly trichome formation and cell wall biogenesis. Importantly, this work sheds light on the mechanisms underlying enhanced resistance to drought and super-infection by fungal pathogens provided by fungal endophyte infection. Finally, our study reveals that not all beneficial plant-microbe associations behave the same in terms of their effects on the host., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi.

Beneficial plant-fungal interactions play an important role in the ability of plants to survive changing environmental conditions. In contrast, phytopathogenic fungi fall at the opposite end of the symbiotic spectrum, causing reduced host growth or even death. In order to exploit beneficial interactions and prevent pathogenic ones, it is essential to understand the molecular differences underlying these alternative states. The association between the endophyte Epichloë festucae and Lolium perenne (perennial ryegrass) is an excellent system for studying these molecular patterns due to the existence of several fungal mutants that have an antagonistic rather than a mutualistic interaction with the host plant. By comparing gene expression in a wild-type beneficial association with three mutant antagonistic associations disrupted in key signaling genes, we identified a core set of 182 genes that show common differential expression patterns between these two states. These gene expression changes are indicative of a nutrient-starvation response, as supported by the upregulation of genes encoding degradative enzymes, transporters, and primary metabolism, and downregulation of genes encoding putative small-secreted proteins and secondary metabolism. These results suggest that disruption of a mutualistic symbiotic interaction may lead to an elevated uptake and degradation of host-derived nutrients and cell-wall components, reminiscent of phytopathogenic interactions.

Epichloë festucae is a mutualistic symbiont that systemically colonizes the intercellular spaces of Lolium perenne leaves to form a highly structured and interconnected hyphal network. In an Agrobacterium tumefaciens T-DNA forward genetic screen, we identified a mutant TM1066 that had a severe host interaction phenotype, causing stunting and premature senescence of the host. Molecular analysis revealed that the mutation responsible for this phenotype was in the cell-wall integrity (CWI) mitogen-activated protein kinase kinase (MAPKK), mkkA. Mutants generated by targeted deletion of the mkkA or the downstream mpkA kinase recapitulated the phenotypes observed for TM1066. Both mutants were defective in hyphal cell–cell fusion, formed intrahyphal hyphae, had enhanced conidiation, and showed microcyclic conidiation. Transmission electron microscopy and confocal microscopy analysis of leaf tissue showed that mutant hyphae were more abundant than the wild type in the intercellular spaces and colonized the vascular bundles. Hyphal branches failed to fuse but, instead, grew past one another to form bundles of convoluted hyphae. Mutant hyphae showed increased fluorescence with AF488-WGA, indicative of increased accessibility of chitin, a hypothesis supported by changes in the cell-wall ultrastructure. These results show that the CWI MAPK pathway is a key signaling pathway for controlling the mutualistic symbiotic interaction between E. festucae and L. perenne.

Transformation is an essential tool for modern fungal research and has played a fundamental role in gaining insight into gene function. Polyethylene glycol (PEG)-mediated transformation of protoplasts is the most commonly used method for genetic transformation of filamentous fungi. Selectable marker genes, that confer resistance to antibiotics, are generally incorporated with the DNA of interest, allowing transformed cells to grow through the antibiotic overlay. Colonies arising from transformed fungal cells are sub-cultured and further analysed. However, the morphological state of the fungal cells during the transformation procedure has been largely overlooked. We investigated the morphological appearance of transformed fungal cells prior to their emergence through the antibiotic overlay. Hyphae appeared to segment and bulge, reminiscent of arthroconidia, an asexual spore typically produced by segmentation of pre-existing hyphae. Selective expression of eGFP under the control of a spore specific promoter, PcatA, in these cells confirmed their spore-like nature. Reducing the oxygen availability to surface-grown cultures partially recapitulated this morphological form. A GFP fusion to the cell wall integrity MAP kinase MpkA localised to the arthroconidia nuclei suggesting the cell wall integrity signalling pathway modulates cell wall stress responses in arthroconidia. This dramatic morphological change was also observed in transformed Magnaporthe oryzae cells suggesting it may be a more general phenomenon in filamentous fungi. Given the changes in cellular structure and spore-like appearance, these observations may have technical implications for deleting genes involved in these processes in Epichloë festucae and, more broadly, a range of fungal species., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

Heterotrimeric (αβγ) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Gα subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as RIC8, has emerged as an important mechanism for Gα regulation in animals. RIC8 is present in animals and filamentous fungi, such as the model eukaryote Neurospora crassa, but is absent from the genomes of baker's yeast and plants. In Neurospora, deletion of ric8 leads to profound defects in growth and asexual and sexual development, similar to those observed for a mutant lacking the Gα genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Δric8 mutants. Similar to reports in Drosophila, Neurospora Δric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Δric8 mutants and suppression of Δric8 by a mutation in the protein kinase A regulatory subunit. RIC8 acts as a GEF for GNA-1 and GNA-3 in vitro, with the strongest effect on GNA-3. Our results support a role for RIC8 in regulating GNA-1 and GNA-3 in Neurospora.

Symbioses between cool season grasses and fungi of the family Clavicipitaceae are an integral component of both natural and agricultural ecosystems. An excellent experimental model is the association between the biotrophic fungus Epichloë festucae and Lolium perenne (perennial ryegrass). The fungal partner produces a suite of secondary metabolites that protect the host from various biotic and abiotic stresses. The plant host provides a source of nutrients and a mechanism of dissemination via seed transmission. Crucial mechanisms that maintain a stable mutualistic association include signaling through the stress activated MAP kinase pathway and production of reactive oxygen species by the fungal NADPH oxidase (Nox) complex. Disruption of components of the Nox complex (NoxA, NoxR and RacA), or the stress-activated MAP kinase (SakA), leads to a breakdown in this finely balanced association, resulting in pathogenic infection instead of mutualism. Hosts infected with fungi lacking a functional Nox complex, or the stress-activated MAP kinase, display a stunted phenotype and undergo premature senescence, while the fungus switches from restricted to proliferative growth. To gain insight into the mechanisms that underlie these physiological changes, high throughput mRNA sequencing has been used to analyze the transcriptomes of both host and symbiont in wild-type and a mutant association. In the ΔsakA mutant association, a dramatic up-regulation of fungal hydrolases and transporters was observed, changes consistent with a switch from restricted symbiotic to proliferative pathogenic growth. Analysis of the plant transcriptome revealed dramatic changes in expression of host genes involved in pathogen defense, transposon activation and hormone biosynthesis and response. This review highlights how finely tuned grass-endophyte associations are, and how interfering with the signaling pathways involved in maintenance of these associations can trigger a change from mutualistic to pathogenic interaction., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Plant-fungal symbioses are a common feature in nature. They vary from pathogenic interactions, where fungi subvert plant resources for their own use, to mutualistic associations, where both fungus and host benefit from the interaction. Although the ecological importance of plant-fungal symbioses has long been recognized and the biology of several key associations are now well studied, new technologies have the potential to allow fresh insight into the molecular basis of plant-fungal interactions. One such technique - high throughput RNA sequencing - has recently been used to explore the molecular basis of cross-species communications. Here, we give a brief overview of this emerging technology, and present a general guide for employing the methodology to dissect plant-fungal symbiosis.

Symbiotic associations between plants and fungi are a dominant feature of many terrestrial ecosystems, yet relatively little is known about the signaling, and associated transcriptome profiles, that define the symbiotic metabolic state. Using the Epichloë festucae-perennial ryegrass (Lolium perenne) association as a model symbiotic experimental system, we show an essential role for the fungal stress-activated mitogen-activated protein kinase (sakA) in the establishment and maintenance of this mutualistic interaction. Deletion of sakA switches the fungal interaction with the host from mutualistic to pathogenic. Infected plants exhibit loss of apical dominance, premature senescence, and dramatic changes in development, including the formation of bulb-like structures at the base of tillers that lack anthocyanin pigmentation. A comparison of the transcriptome of wild-type and sakA associations using high-throughput mRNA sequencing reveals dramatic changes in fungal gene expression consistent with the transition from restricted to proliferative growth, including a down-regulation of several clusters of secondary metabolite genes and up-regulation of a large set of genes that encode hydrolytic enzymes and transporters. Analysis of the plant transcriptome reveals up-regulation of host genes involved in pathogen defense and transposon activation as well as dramatic changes in anthocyanin and hormone biosynthetic/responsive gene expression. These results highlight the fine balance between mutualism and antagonism in a plant-fungal interaction and the power of deep mRNA sequencing to identify candidate sets of genes underlying the symbiosis.

Regulated synthesis of reactive oxygen species (ROS) by specific fungal NADPH oxidases (Noxs) plays a key role in fungal cellular differentiation and development. Fungi have up to three different Nox isoforms, NoxA, B and C. The NoxA isoform has a key role in triggering the development of fruiting bodies in several sexual species whereas NoxB plays a key role in ascospore germination. The function of NoxC remains unknown. Both NoxA and NoxB are required for the development of fungal infection structures by some plant pathogens. ROS production by NoxA is critical for maintaining a fungal-plant symbiosis. Localised synthesis of ROS is also important in establishing and maintaining polarised hyphal growth. Activation of NoxA/NoxB requires the regulatory subunit, NoxR, and the small GTPase RacA. The BemA scaffold protein may also be involved in the assembly of the Nox complex. By analogy with mammalian systems MAP and PAK kinases may regulate fungal Nox activation. How fungal cells sense and respond to ROS associated with cellular differentiations remains to be discovered.

The ability of fungi to sense and respond rapidly to environmental stress is crucial for their survival in the wild. One of the most important pathways involved in this response is the stress-activated MAP (mitogen-activated protein) kinase pathway. We report here on the isolation of the stress-activated MAP kinase, sakA, from the fungal endophyte Epichloë festucae. Complementation of the stress sensitivity and cell cycle defects of an Schizosaccharomyces pombe sty1Delta mutant with sakA confirmed it encodes a functional MAP kinase. Analysis of an E. festucae DeltasakA mutant revealed sakA is essential for growth under conditions of temperature and osmotic stress in culture, and for sensitivity to the fungicide fludioxonil. However, the DeltasakA mutant shows no increased sensitivity to hydrogen peroxide. Given sakA can rescue the sty1Delta mutant from sensitivity to oxidative stress, SakA has the potential to sense and transduce oxidative stress signals. The DeltasakA mutant is also defective in conidia formation, suggesting a role for SakA in asexual development of E. festucae. The detection of elevated hydrogen peroxide production in the DeltasakA mutant suggests there may be a link between MAP kinase and ROS (reactive oxygen species) signalling pathways in E. festucae.

Unlabelled: Avulsion injuries have a poor prognosis for survival in clinical replantation surgery. Arterial thrombosis is the most significant factor contributing to avulsion replant failure, and severe arterial damage has been observed with this injury. However, patency rates of experimentally avulsed arteries repaired immediately are much higher than in the clinical situation. This paper evaluates the effect of an added component--ischemia--on the patency of experimentally avulsed arteries. All avulsions seen clinically are subject to some degree of ischemia prior to replantation. Ninety rabbits had both femoral arteries avulsed under general anesthesia. A 6.5-cm graft was harvested from the left distal femoral artery. In 20 rabbits (group 1: 0 hours of ischemia) the graft was immediately inserted into the defect in the right femoral artery. Sixty rabbits (20 grafts per group) had their grafts stored at 4 degrees C for either 10 hours (group 2), 18 hours (group 3), or 24 hours (group 4) and reinserted into the right femoral artery in a second operation. Patency was assessed 3 weeks after reinsertion. Groups 1 and 2 maintained high patency rates (85 percent); however, group 3 (70 percent) and group 4 (45 percent) had lower patency rates than group 1, with a significant difference between groups 1 and 4 (p < 0.01). In a fifth group (10 grafts), avulsed 24-hour ischemic grafts were hydrodilated prior to reinsertion. The patency rate of this group increased significantly (90 percent) compared with group 4 (p < 0.005)., Conclusion: These experiments suggest that a combination of avulsion injury and ischemia time is responsible for the poor clinical results of avulsion replantations.

The objective of this study was to determine the effects of repetitive motion on the flexor tendons and synovium in the carpal tunnel. One possible mechanism of tendon damage is fraying of the tendons as they slide past each other in the carpal tunnel. Increases in tendon friction were measured in human cadaver arms. The flexor tendons were loaded using pneumatic cylinders while load cells were used to measure tendon force both distal and proximal to the carpal tunnel. Results showed that tendon force distal to the carpal tunnel decreased by over 10% after 6 h, while tendon force proximal to the carpal tunnel remained constant. Two rhesus monkeys were used to further study the effects of repetitive motion. One arm of each animal was subjected to 729000 repetitive cycles over a 3-week period. Results of the animal study showed that highly repetitive motions encountered over a relatively short period of time had little effect on the tissue in and around the carpal tunnel., (Copyright © 1994. Published by Elsevier Ltd.)

Symptomatic radial nerve compression is relatively uncommon. A relatively high incidence of compressive neuropathy involves other major nerves in the same extremity. Because sensory complaints are minor, radial nerve compression may successfully masquerade as tendonitis or tendon rupture. The most common site of radial nerve compression is in the forearm, at the arcade of Frohse. Spontaneous onset of dense paralysis is often due to space-occupying lesions in the forearm. Trauma-related compression in the forearm is most often due to radial head dislocation and either humeral fracture or local external pressure in the upper arm. Compression resulting in weakness that does not improve with several months of splinting, anti-inflammatory medication, and activity changes should be treated surgically to reduce the extent of permanent deficit. After decompression, early active motion is instituted to encourage nerve gliding. Results after decompression are not as favorable as those for carpal or cubital tunnel release. The worst results of decompression are seen in patients who have work-related injuries, chronic pain, and poor localization of symptoms on physical examination.

Many options exist to provide soft-tissue cover of the thumb. The choice of cover should be dictated by the defect itself: the location, size, and requirements for sensibility and durability. Within these boundaries, the choice of cover should be in accordance with the goals of any extremity reconstruction, permitting primary wound healing and early active use.

Toe-to-hand transfer is a well-established reconstructive option for certain congenital hand anomalies. It is the only technique which can add growth potential to the immature skeleton. Toe transfer is best suited for constriction ring amputations, which have relatively normal proximal anatomy. Transfers should be performed early in life to avoid lack of cortical integration of the new part. Anatomic variations of both hand and foot are often encountered, which influence both operative approach and functional prognosis. Indications, techniques, and complications are reviewed.

Congenital syndactyly is associated with a wide spectrum of local anomalies that strongly influence the ultimate outcome of treatment. Surgical options, indications, timing, and prognosis are all determined by the preoperative degree of deformity. The surgeon must apply knowledge of anatomic variations and adhere to fundamental principles of technique, which are reviewed in detail.

The efficacy of transferring vascularised lymph nodes into lymphoedematous limbs was investigated. Stable below-knee lymphoedema was established in one hind limb of 10 dogs. The superficial inguinal lymph node and perinodal tissue from the normal hind limb was moved to the popliteal region of the lymphoedematous leg as a free vascularised transfer. In five dogs lymphaticolymphatic anastomoses between lymphatics of the node and proximal recipient site lymphatics were also performed. Circumferential measurements of the foot, ankle and midleg were obtained preoperatively and postoperatively at 3 and 6 months. These measurements showed postoperative reduction of the lymphoedematous legs compared to controls, with no added benefit from lymphaticolymphatic anastomoses. Technetium 99 scans and lymphangiography demonstrated re-establishment of lymphatic continuity in all recipient legs at 3 and 6 months post-transfer. Histological examination at 3 and 6 months revealed normal architecture in 9 of 10 nodes, although areas of lymphocyte depletion were common. Vascularised lymph node transfer to a lymphoedematous leg re-established lymphatic continuity and resulted in partial reduction of limb size. The addition of lymphaticolymphatic anastomosis to vascularized node transfer is neither necessary nor beneficial.

An outpatient diagnositc technique comparable to the Papanicolaou smear for carcinoma of the cervix is needed to permit early recognition of adenocarcinoma of the endometrium. Five different techniques of sampling the endometrial cavity were carried out on 1, 742 patients at the University of Michigan Medical Center. A total of 2,553 specimens were correlated with operating room curettings to assess the accuracy of each sampling technique. The methods of saline irrigation with an antrum cannula, endometrial brush sampling, high-vacuum aspiration, and the Gravlee jet washer proved not to be accurate enough to be used as a routine screeening test. There was excellent correlation (96 per cent) between endometrial biopsies and curettings. In our experience, endometrial biopsy with a suction or basket curette was the diagnostic technique short of curettage which permitted accurate diagnosis.