Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility.

BACKGROUND: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). METHODS: We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. RESULTS: iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators. CONCLUSIONS: iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

An integrated in vitro carcinogenicity test that distinguishes between genotoxic carcinogens, non-genotoxic carcinogens, and non-carcinogens.

Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.

Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, ß- and γ- TLO gene function.

The Candida albicans genome contains between ten and fifteen distinct TLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo in C. albicans we deleted all fourteen TLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in the tloΔ mutant strain compared to the parent, while RNA-seq analysis showed that the tloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, the tloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, ß- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees. TLOα1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e., TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression of TLOß2 in the tloΔ mutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloß2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegrated TLO genes in a tloΔ/med3Δ double mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range of C. albicans cellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.

Evaluation of endpoints for the study and diagnosis of mitochondrial toxicity and disease: a narrative review.

Mitochondrial DNA mutation and toxicity have been linked to several inherited and acquired diseases; however, these are challenging to diagnose and characterize due to clinical and genetic heterogeneity. This review investigates current techniques for the analysis of mitochondrial perturbations, and novel, emerging endpoints for routine application within the clinical setting. Particular focus is given to the biochemistry of the mitochondria influencing each endpoint and the relation of these to toxicity. Current approaches such as the use of metabolic markers (e.g. lactate production), and muscle biopsies to measure mitochondrial proteins were found to lack specificity. Newly emerging identified endpoints were: fibroblast growth factor-21, glucose uptake, mitochondrial membrane potential, mitochondrial morphology, mtDNA heteroplasmy, and mutation of mtDNA and nuclear DNA. Owed to the advancement in genetic analysis techniques, it is suggested by this review that genotypic endpoints of mtDNA mutation and heteroplasmy show particular promise as indicators of mitochondrial disease. It is, however, acknowledged that any single endpoint in isolation offers limited information; therefore, it is recommended that analysis of several endpoints simultaneously will offer the greatest benefit in terms of disease diagnosis and study. It is hoped that this review further highlights the need for advancement in understanding mitochondrial disease.

Early microglial response, myelin deterioration and lethality in mice deficient for very long chain ceramide synthesis in oligodendrocytes.

The sphingolipids galactosylceramide (GalCer), sulfatide (ST) and sphingomyelin (SM) are essential for myelin stability and function. GalCer and ST are synthesized mostly from C22-C24 ceramides, generated by Ceramide Synthase 2 (CerS2). To clarify the requirement for C22-C24 sphingolipid synthesis in myelin biosynthesis and stability, we generated mice lacking CerS2 specifically in myelinating cells (CerS2ΔO/ΔO ). At 6 weeks of age, normal-appearing myelin had formed in CerS2ΔO/ΔO mice, however there was a reduction in myelin thickness and the percentage of myelinated axons. Pronounced loss of C22-C24 sphingolipids in myelin of CerS2ΔO/ΔO mice was compensated by greatly increased levels of C18 sphingolipids. A distinct microglial population expressing high levels of activation and phagocytic markers such as CD64, CD11c, MHC class II, and CD68 was apparent at 6 weeks of age in CerS2ΔO/ΔO mice, and had increased by 10 weeks. Increased staining for denatured myelin basic protein was also apparent in 6-week-old CerS2ΔO/ΔO mice. By 16 weeks, CerS2ΔO/ΔO mice showed pronounced myelin atrophy, motor deficits, and axon beading, a hallmark of axon stress. 90% of CerS2ΔO/ΔO mice died between 16 and 26 weeks of age. This study highlights the importance of sphingolipid acyl chain length for the structural integrity of myelin, demonstrating how a modest reduction in lipid chain length causes exposure of a denatured myelin protein epitope and expansion of phagocytic microglia, followed by axon pathology, myelin degeneration, and motor deficits. Understanding the molecular trigger for microglial activation should aid the development of therapeutics for demyelinating and neurodegenerative diseases.

Remodelling of myelinated axons and oligodendrocyte differentiation is stimulated by environmental enrichment in the young adult brain.

Oligodendrocyte production and myelination continues lifelong in the central nervous system (CNS), and all stages of this process can be adaptively regulated by neuronal activity. While artificial exogenous stimulation of neuronal circuits greatly enhances oligodendrocyte progenitor cell (OPC) production and increases myelination during development, the extent to which physiological stimuli replicates this is unclear, particularly in the adult CNS when the rate of new myelin addition slows. Here, we used environmental enrichment (EE) to physiologically stimulate neuronal activity for 6 weeks in 9-week-old C57BL/six male and female mice and found no increase in compact myelin in the corpus callosum or somatosensory cortex. Instead, we observed a global increase in callosal axon diameter with thicker myelin sheaths, elongated paranodes and shortened nodes of Ranvier. These findings indicate that EE induced the dynamic structural remodelling of myelinated axons. Additionally, we observed a global increase in the differentiation of OPCs and pre-myelinating oligodendroglia in the corpus callosum and somatosensory cortex. Our findings of structural remodelling of myelinated axons in response to physiological neural stimuli during young adulthood provide important insights in understanding experience-dependent myelin plasticity throughout the lifespan and provide a platform to investigate axon-myelin interactions in a physiologically relevant context.

Generation and characterisation of four multiple sclerosis iPSC lines from a single family.

Multiple sclerosis (MS) is a complex neuroinflammatory/degenerative disease of the central nervous system (CNS) that results in the formation of demyelinated lesions and axon degeneration. MS aetiology is complex, with genetics estimated to account for ∼48% of MS risk (International Multiple Sclerosis Genetics Consortium, 2019). Despite this, families with a high incidence of MS are rare. We have generated four induced pluripotent stem cell (iPSC) lines from individuals with relapsing-remitting and secondary progressive MS within a single family. The generation of disease-specific iPSC lines from multiple members of a single family will facilitate MS genetic and functional studies.

Using MS induced pluripotent stem cells to investigate MS aetiology.

Multiple sclerosis (MS) is a complex disease, and its pathophysiology impacts the function of immune and central nervous system cell types. Despite extensive investigation into the aetiology of MS, the underlying cause/s remain elusive and consequently, faithful in vitro or in vivo preclinical models of MS do not exist. Advances in human stem cell technologies have enabled the generation of induced pluripotent stem cells (iPSCs) from people with MS. This review summarises the discoveries made using iPSCs derived from people with MS and explores their current and potential application/s in MS research.

Effectiveness of caregiver interventions for people with cancer and non-cancer-related chronic pain: a systematic review and meta-analysis.

BACKGROUND: People with chronic pain frequently have difficulties in completing everyday tasks to maintain independence and quality of life. Informal caregivers may provide support to these individuals. However, the effectiveness of interventions to train and support these individuals in caregiving remains unclear. The purpose of this study was to systematically review the evidence to determine the effectiveness of caregiver interventions to support informal caregivers of people with chronic pain. METHODS: A systematic review of published and unpublished literature databases was undertaken (9 April 2021). Trials reporting clinical outcomes of caregiver interventions to train informal caregivers to support community-dwelling people with chronic pain were included. Meta-analysis was undertaken and each outcome was assessed using Grading of Recommendations, Assessment, Development and Evaluation. RESULTS: Twenty-seven studies were eligible (N = 3427 patients). Twenty-four studies assessed patients with cancer pain and three with musculoskeletal pain. No other patient groups were identified. There was very low-quality evidence that caregiver interventions were beneficial for caregiver health-related quality of life (standardised mean difference = 0.26, 95% confidence interval = 0.01 to 0.52; N = 231). There was moderate-quality evidence that caregiving interventions were effective in reducing pain in the short-term (standardised mean difference = 0.16, 95% confidence interval = -0.29 to -0.03). There was low-quality evidence that caregiving interventions had no beneficial effect over usual care for psychological outcomes, fatigue, coping or physical function in the long-term. CONCLUSION: Caregiving interventions may be effective for patients and caregivers but only in the shorter-term and for a limited number of outcomes. There is insufficient evidence examining the effectiveness of caregiver interventions for people with non-cancer-related pain.

Lived experiences of informal caregivers of people with chronic musculoskeletal pain: a systematic review and meta-ethnography.

BACKGROUND: People with chronic pain often seek support from friends and family for everyday tasks. These individuals are termed informal caregivers. There remains uncertainty regarding the lived experiences of these people who care for individuals with chronic musculoskeletal pain. The aim of this article is to synthase the evidence on the lived experiences of informal caregivers providing care to people with chronic musculoskeletal pain. METHODS: A systematic literature review was undertaken of published and unpublished literature databases including EMBASE, MEDLINE, CINAHL, PubMed, the WHO International Clinical Trial Registry and ClinicalTrials.gov registry (to September 2019). Qualitative studies exploring the lived experiences of informal caregivers of people with chronic musculoskeletal pain were included. Data were synthesised using a meta-ethnography approach. Evidence was evaluated using the Critical Appraisal Skills Programme qualitative appraisal tool. RESULTS: From 534 citations, 10 studies were eligible (360 participants: 171 informal caregivers of 189 care recipients). The evidence was moderate quality. Seven themes arose: the relationship of caregivers to healthcare professionals; role reversal with care recipients; acting the confidant to the care recipient; a constant burden in caregiving; legitimising care recipient's condition; knowledge and skills to provide caregiving; and the perception of other family members and wider society to the caregiver/care recipient dyad. CONCLUSION: The lived experiences of caregivers of people with chronic musculoskeletal pain is complex and dynamic. There is an inter-connected relationship between caregivers, care recipients and healthcare professionals. Exploring how these experiences can be modified to improve a caregiving dyad's lived experience is now warranted.

Oligodendrogenesis and myelination regulate cortical development, plasticity and circuit function.

During cortical development and throughout adulthood, oligodendrocytes add myelin internodes to glutamatergic projection neurons and GABAergic inhibitory neurons. In addition to directing node of Ranvier formation, to enable saltatory conduction and influence action potential transit time, oligodendrocytes support axon health by communicating with axons via the periaxonal space and providing metabolic support that is particularly critical for healthy ageing. In this review we outline the timing of oligodendrogenesis in the developing mouse and human cortex and describe the important role that oligodendrocytes play in sustaining and modulating neuronal function. We also provide insight into the known and speculative impact that myelination has on cortical axons and their associated circuits during the developmental critical periods and throughout life, particularly highlighting their life-long role in learning and remembering.

Acute treatment with TrkB agonist LM22A-4 confers neuroprotection and preserves myelin integrity in a mouse model of pediatric traumatic brain injury.

Young children have a high risk of sustaining a traumatic brain injury (TBI), which can have debilitating life-long consequences. Importantly, the young brain shows particular vulnerability to injury, likely attributed to ongoing maturation of the myelinating nervous system at the time of insult. Here, we examined the effect of acute treatment with the partial tropomyosin receptor kinase B (TrkB) agonist, LM22A-4, on pathological and neurobehavioral outcomes after pediatric TBI, with the hypothesis that targeting TrkB would minimize tissue damage and support functional recovery. We focused on myelinated tracts-the corpus callosum and external capsules-based on recent evidence that TrkB activation potentiates oligodendrocyte remyelination. Male mice at postnatal day 21 received an experimental TBI or sham surgery. Acutely post-injury, extensive cell death, a robust glial response and disruption of compact myelin were evident in the injured brain. TBI or sham mice then received intranasal saline vehicle or LM22A-4 for 14 days. Behavior testing was performed from 4 weeks post-injury, and brains were collected at 5 weeks for histology. TBI mice showed hyperactivity, reduced anxiety-like behavior, and social memory impairments. LM22A-4 ameliorated the abnormal anxiolytic phenotype but had no effect on social memory deficits. Use of spectral confocal reflectance microscopy detected persistent myelin fragmentation in the external capsule of TBI mice at 5 weeks post-injury, which was accompanied by regionally distinct deficits in oligodendrocyte progenitor cells and post-mitotic oligodendrocytes, as well as chronic reactive gliosis and atrophy of the corpus callosum and injured external capsule. LM22A-4 treatment ameliorated myelin deficits in the perilesional external capsule, as well as tissue volume loss and the extent of reactive gliosis. However, there was no effect of this TrkB agonist on oligodendroglial populations detected at 5 weeks post-injury. Collectively, our results demonstrate that targeting TrkB immediately after TBI during early life confers neuroprotection and preserves myelin integrity, and this was associated with some improved neurobehavioral outcomes as the pediatric injured brain matures.

Barriers to uptake of the hip fracture core outcome set: An international survey of 80 hip fracture trialists.

BACKGROUND: Core outcome sets are an agreed recommendation to inform the selection of outcome measures in clinical trials. There has been low uptake of the 2014 hip fracture core outcome set. The reasons for this remain unclear. The aim of this study was to understand the reasons for the non-adoption and approaches to increase adoption of the hip fracture core outcome set. METHODS: Randomised controlled trials from PubMed (2017-2019) and ClinicalTrials.gov (2015-2019) were identified. Corresponding authors for each identified trial (n = 302) were surveyed using five questions on awareness of the hip fracture core outcome set, reasons for non-adoption and approaches to increase adoption. Data were analysed descriptively using frequencies, mean values and standard deviations. RESULTS: Fifty-four percent of the respondents (n = 43) were aware of the concept of core outcome set. Only 15% (n = 12) based the outcome measure selection on the 2014 hip fracture core outcome set. Key reasons for non-adoption included the following: authors being unaware and perceived inappropriateness to their trial design. Eighty-six percent (n = 69) of respondents agreed to the need for increased awareness of core outcome sets through research training, academic and clinical journal requirements, and funding or publication stipulations. Eighty-eight percent (n = 70) of respondents indicated the current core outcome set required revision to focus on trials investigating people with cognitive impairment, caregivers, rehabilitation, surgical interventions and anaesthetic trial designs. CONCLUSION: Barriers to the adoption of the hip fracture core outcome set centre on education, awareness of the core outcome sets and applicability to the breath of hip fracture trial designs. Further consideration should be made to address these, to improve the harmonisation of outcome measures across hip fracture trials.

Imaging and Quantification of Myelin Integrity After Injury With Spectral Confocal Reflectance Microscopy.

Developing a high-throughput approach to quantify the extent of myelin integrity in preclinical models of demyelinating diseases will enhance our capacity to identify novel therapies for myelin repair. In light of the technical limitations of electron microscopy and immunohistochemical analyses of myelination, we have utilized a novel imaging technique, spectral confocal reflectance (SCoRe) microscopy. SCoRe takes advantage of the optically reflective properties of compact myelin, allowing the integrity of compact myelin to be quantified over the course of the cuprizone-induced model of central demyelination. We applied SCoRe imaging on fixed frozen brain sections. SCoRe analysis of control mice identified an increase in corpus callosum myelination during the period of cuprizone administration and recovery, suggesting that the normal developmental processes of myelination are ongoing at this time. Importantly, analysis of mice subjected to cuprizone identified a significant reduction in compact myelin in both rostral and caudal corpus callosum compared to age-matched control mice. SCoRe microscopy also allowed the visualization and quantification of the amount of myelin debris in demyelinating lesions. Combining SCoRe imaging with immunohistochemistry, we quantified the amount of myelin debris within IBA-1+ microglia and found that 11% of myelin debris colocalized in microglia irrespective of the callosal regions, with the vast majority of debris outside of microglia. In summary, we have demonstrated that SCoRe microscopy is an effective and powerful tool to perform both quantitative and qualitative analyses of compact myelin integrity in health or after injury in vivo, demonstrating its future application in high-throughput assessments and screening of the therapeutic efficacy of myelin repair therapies in preclinical animal models of demyelinating diseases.

TrkB Agonist LM22A-4 Increases Oligodendroglial Populations During Myelin Repair in the Corpus Callosum.

The neurotrophin, brain-derived neurotrophic factor (BDNF) promotes central nervous system (CNS) myelination during development and after injury. This is achieved via activation of oligodendrocyte-expressed tropomyosin-related kinase (Trk) B receptors. However, while administration of BDNF has shown beneficial effects, BDNF itself has a poor pharmacokinetic profile. Here, we compare two TrkB-targeted BDNF-mimetics, the structural-mimetic, tricyclic dimeric peptide-6 (TDP6) and the non-peptide small molecule TrkB agonist LM22A-4 in a cuprizone model of central demyelination in female mice. Both mimetics promoted remyelination, increasing myelin sheath thickness and oligodendrocyte densities after 1-week recovery. Importantly, LM22A-4 exerts these effects in an oligodendroglial TrkB-dependent manner. However, analysis of TrkB signaling by LM22A-4 suggests rather than direct activation of TrkB, LM22A-4 exerts its effects via indirect transactivation of Trk receptors. Overall, these studies support the therapeutic strategy to selectively targeting TrkB activation to promote remyelination in the brain.

Inhibiting Bone Morphogenetic Protein 4 Type I Receptor Signaling Promotes Remyelination by Potentiating Oligodendrocyte Differentiation.

Blocking inhibitory factors within CNS demyelinating lesions is regarded as a promising strategy to promote remyelination. Bone morphogenetic protein 4 (BMP4) is an inhibitory factor present in demyelinating lesions. Noggin, an endogenous antagonist to BMP, has previously been shown to increase the number of oligodendrocytes and promote remyelination in vivo. However, it remains unclear how BMP4 signaling inhibits remyelination. Here we investigated the downstream signaling pathway that mediates the inhibitory effect that BMP4 exerts upon remyelination through pharmacological and transgenic approaches. Using the cuprizone mouse model of central demyelination, we demonstrate that selectively blocking BMP4 signaling via the pharmacological inhibitor LDN-193189 significantly promotes oligodendroglial differentiation and the extent of remyelination in vivo This was accompanied by the downregulation of transcriptional targets that suppress oligodendrocyte differentiation. Further, selective deletion of BMP receptor type IA (BMPRIA) within primary mouse oligodendrocyte progenitor cells (OPCs) significantly enhanced their differentiation and subsequent myelination in vitro Together, the results of this study identify that BMP4 signals via BMPRIA within OPCs to inhibit oligodendroglial differentiation and their capacity to myelinate axons, and suggest that blocking the BMP4/BMPRIA pathway in OPCs is a promising strategy to promote CNS remyelination.

Brain-Derived Neurotrophic Factor in Central Nervous System Myelination: A New Mechanism to Promote Myelin Plasticity and Repair.

Brain-derived neurotrophic factor (BDNF) plays vitally important roles in neural development and plasticity in both health and disease. Recent studies using mutant mice to selectively manipulate BDNF signalling in desired cell types, in combination with animal models of demyelinating disease, have demonstrated that BDNF not only potentiates normal central nervous system myelination in development but enhances recovery after myelin injury. However, the precise mechanisms by which BDNF enhances myelination in development and repair are unclear. Here, we review some of the recent progress made in understanding the influence BDNF exerts upon the myelinating process during development and after injury, and discuss the cellular and molecular mechanisms underlying its effects. In doing so, we raise new questions for future research.

Expansion of the TLO gene family enhances the virulence of Candida species.

The TLO genes are a family of subtelomeric ORFs in the fungal pathogens Candida albicans and C. dubliniensis encoding a subunit of the Mediator complex homologous to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two. To investigate if expansion of the TLO repertoire in C. dubliniensis has an effect on phenotype and virulence we expressed three representative C. albicans TLO genes (TLOß2, TLOγ11 and TLOα12) in a wild type C. dubliniensis background, under the control of either their native or the ACT1 promoter. Expression of TLOß2 resulted in a hyperfilamentous phenotype, while overexpression of TLOγ11 and TLOα12 resulted in enhanced resistance to oxidative stress. Expression of all three TLO genes from the ACT1 promoter resulted in increased virulence in the Galleria infection model. In order to further investigate if individual TLO genes exhibit differences in function we expressed six representative C. albicans TLO genes in a C. dubliniensis Δtlo1/Δtlo2 double mutant. Differences were observed in the ability of the expressed CaTLOs to complement the various phenotypes of the mutant. All TLO genes with the exception of TLOγ7 could restore filamentation, however only TLOα9, γ11 and α12 could restore chlamydospore formation. Differences in the ability of CaTLO genes to restore growth in the presence of H2O2, calcofluor white, Congo red and at 42°C were observed. Only TLOα3 restored wild-type levels of virulence in the Galleria infection model. These data show that expansion of the TLO gene family in C. dubliniensis results in gain of function and that there is functional diversity amongst members of the gene family. We propose that this expansion of the TLO family contributes to the success of C. albicans as a commensal and opportunistic pathogen.