A randomised, double blind, placebo-controlled trial of a two-week course of dexamethasone for adult patients with a symptomatic Chronic Subdural Haematoma (Dex-CSDH trial).

Background: Chronic subdural haematoma is a collection of 'old blood' and its breakdown products in the subdural space and predominantly affects older people. Surgical evacuation remains the mainstay in the management of symptomatic cases. Objective: The Dex-CSDH (DEXamethasone in Chronic SubDural Haematoma) randomised trial investigated the clinical effectiveness and cost-effectiveness of dexamethasone in patients with a symptomatic chronic subdural haematoma. Design: This was a parallel, superiority, multicentre, pragmatic, randomised controlled trial. Assigned treatment was administered in a double-blind fashion. Outcome assessors were also blinded to treatment allocation. Setting: Neurosurgical units in the UK. Participants: Eligible participants included adults (aged ≥ 18 years) admitted to a neurosurgical unit with a symptomatic chronic subdural haematoma confirmed on cranial imaging. Interventions: Participants were randomly assigned in a 1 : 1 allocation to a 2-week tapering course of dexamethasone or placebo alongside standard care. Main outcome measures: The primary outcome was the Modified Rankin Scale score at 6 months dichotomised to a favourable (score of 0-3) or an unfavourable (score of 4-6) outcome. Secondary outcomes included the Modified Rankin Scale score at discharge and 3 months; number of chronic subdural haematoma-related surgical interventions undertaken during the index and subsequent admissions; Barthel Index and EuroQol 5-Dimension 5-Level utility index score reported at discharge, 3 months and 6 months; Glasgow Coma Scale score reported at discharge and 6 months; mortality at 30 days and 6 months; length of stay; discharge destination; and adverse events. An economic evaluation was also undertaken, during which the net monetary benefit was estimated at a willingness-to-pay threshold of £20,000 per quality-adjusted life-year. Results: A total of 748 patients were included after randomisation: 375 were assigned to dexamethasone and 373 were assigned to placebo. The mean age of the patients was 74 years and 94% underwent evacuation of their chronic subdural haematoma during the trial period. A total of 680 patients (91%) had 6-month primary outcome data available for analysis: 339 in the placebo arm and 341 in the dexamethasone arm. On a modified intention-to-treat analysis of the full study population, there was an absolute reduction in the proportion of favourable outcomes of 6.4% (95% confidence interval 11.4% to 1.4%; p = 0.01) in the dexamethasone arm compared with the control arm at 6 months. At 3 months, the between-group difference was also in favour of placebo (-8.2%, 95% confidence interval -13.3% to -3.1%). Serious adverse events occurred in 60 out of 375 (16.0%) in the dexamethasone arm and 24 out of 373 (6.4%) in the placebo arm. The net monetary benefit of dexamethasone compared with placebo was estimated to be -£97.19. Conclusions: This trial reports a higher rate of unfavourable outcomes at 6 months, and a higher rate of serious adverse events, in the dexamethasone arm than in the placebo arm. Dexamethasone was also not estimated to be cost-effective. Therefore, dexamethasone cannot be recommended for the treatment of chronic subdural haematoma in this population group. Future work and limitations: A total of 94% of individuals underwent surgery, meaning that this trial does not fully define the role of dexamethasone in conservatively managed haematomas, which is a potential area for future study. Trial registration: This trial is registered as ISRCTN80782810. Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 13/15/02) and is published in full in Health Technology Assessment; Vol. 28, No. 12. See the NIHR Funding and Awards website for further award information.

Chronic subdural haematoma is one of the most common conditions managed in adult neurosurgery and mainly affects older people. It is an 'old' collection of blood and blood breakdown products found on the surface of the brain. Surgery to drain the liquid collection is effective, with most patients improving. Given that inflammation is involved in the disease process, a commonly used steroid, dexamethasone, has been used alongside surgery or instead of surgery since the 1970s. However, there is no consensus or high-quality studies confirming the effectiveness of dexamethasone for the treatment of chronic subdural haematoma. This study was designed to determine the effectiveness of adding dexamethasone to the normal treatment for patients with a symptomatic chronic subdural haematoma. The benefit of adding dexamethasone was measured using a disability score called the Modified Rankin Scale, which can be divided into favourable and unfavourable outcomes. This was assessed at 6 months after entry into the study. In total, 748 adults with a symptomatic chronic subdural haematoma treated in neurosurgical units in the UK participated. Each participant had an equal chance of receiving either dexamethasone or a placebo because they were assigned randomly. Neither the patients nor the investigators knew who received dexamethasone and who received placebo. Most patients in both groups had an operation to drain the haematoma and experienced significant functional improvement at 6 months compared with their initial admission to hospital. However, patients who received dexamethasone had a lower chance than patients who received placebo of favourable recovery at 6 months. Specifically, 84% of patients who received dexamethasone had recovered well at 6 months, compared with 90% of patients who received placebo. There were more complications in the group that received dexamethasone. This trial demonstrates that adding dexamethasone to standard treatment reduced the chance of a favourable outcome compared with standard treatment alone. Therefore, this study does not support the use of dexamethasone in treating patients with a symptomatic chronic subdural haematoma.

A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Embers of the Past: Early Childhood Traumas Interact with Variation in P2RX7 Gene Implicated in Neuroinflammation on Markers of Current Suicide Risk.

Both early childhood traumatic experiences and current stress increase the risk of suicidal behaviour, in which immune activation might play a role. Previous research suggests an association between mood disorders and P2RX7 gene encoding P2X7 receptors, which stimulate neuroinflammation. We investigated the effect of P2RX7 variation in interaction with early childhood adversities and traumas and recent stressors on lifetime suicide attempts and current suicide risk markers. Overall, 1644 participants completed questionnaires assessing childhood adversities, recent negative life events, and provided information about previous suicide attempts and current suicide risk-related markers, including thoughts of ending their life, death, and hopelessness. Subjects were genotyped for 681 SNPs in the P2RX7 gene, 335 of which passed quality control and were entered into logistic and linear regression models, followed by a clumping procedure to identify clumps of SNPs with a significant main and interaction effect. We identified two significant clumps with a main effect on current suicidal ideation with top SNPs rs641940 and rs1653613. In interaction with childhood trauma, we identified a clump with top SNP psy_rs11615992 and another clump on hopelessness containing rs78473339 as index SNP. Our results suggest that P2RX7 variation may mediate the effect of early childhood adversities and traumas on later emergence of suicide risk.

Rapid quantification of biological nitrogen fixation using optical spectroscopy.

Biological nitrogen fixation (BNF) provides a globally important input of nitrogen (N); its quantification is critical but technically challenging. Leaf reflectance spectroscopy offers a more rapid approach than traditional techniques to measure plant N concentration ([N]) and isotopes (δ15N). Here we present a novel method for rapidly and inexpensively quantifying BNF using optical spectroscopy. We measured plant [N], δ15N, and the amount of N derived from atmospheric fixation (Ndfa) following the standard traditional methodology using isotope ratio mass spectrometry (IRMS) from tissues grown under controlled conditions and taken from field experiments. Using the same tissues, we predicted the same three parameters using optical spectroscopy. By comparing the optical spectroscopy-derived results with traditional measurements (i.e. IRMS), the amount of Ndfa predicted by optical spectroscopy was highly comparable to IRMS-based quantification, with R2 being 0.90 (slope=0.90) and 0.94 (slope=1.02) (root mean square error for predicting legume δ15N was 0.38 and 0.43) for legumes grown in glasshouse and field, respectively. This novel application of optical spectroscopy facilitates BNF studies because it is rapid, scalable, low cost, and complementary to existing technologies. Moreover, the proposed method successfully captures the dynamic response of BNF to climate changes such as warming and drought.

Chiral and Achiral Crystal Structures of Au25 (PET)18 0 Reveal Effects of Ligand Rotational Isomerization on Optoelectronic Properties.

The crystal structures of 4 ligand-rotational isomers of Au25 (PET)18 are presented. Two new ligand-rotational isomers are revealed, and two higher-quality structures (allowing complete solution of the ligand shell) of previously solved Au25 (PET)18 clusters are also presented. One of the structures lacks an inversion center, making it the first chiral Au25 (SR)18 structure solved. These structures combined with previously published Au25 (SR)18 structures enable an analysis of the empirical ligand conformation landscape for Au25 (SR)18 clusters. This analysis shows that the dihedral angles within the PET ligand are restricted to certain observable values, and also that the dihedral angle values are interdependent, in a manner reminiscent of biomolecule dihedral angles such as those in proteins and DNA. The influence of ligand conformational isomerism on optical and electronic properties was calculated, revealing that the ligand conformations affect the nanocluster absorption spectrum, which potentially provides a way to distinguish between isomers at low temperature.

Fungal metabolism and free amino acid content may predict nitrogen transfer to the host plant in the ectomycorrhizal relationship between Pisolithus spp. and Eucalyptus grandis.

Ectomycorrhizal (ECM) fungi are crucial for tree nitrogen (N) nutrition; however, mechanisms governing N transfer from fungal tissues to the host plant are not well understood. ECM fungal isolates, even from the same species, vary considerably in their ability to support tree N nutrition, resulting in a range of often unpredictable symbiotic outcomes. In this study, we used isotopic labelling to quantify the transfer of N to the plant host by isolates from the ECM genus Pisolithus, known to have significant variability in colonisation and transfer of nutrients to a host. We considered the metabolic fate of N acquired by the fungi and found that the percentage of plant N acquired through symbiosis significantly correlated to the concentration of free amino acids in ECM extra-radical mycelium. Transcriptomic analyses complemented these findings with isolates having high amino acid content and N transfer showing increased expression of genes related to amino acid transport and catabolic pathways. These results suggest that fungal N metabolism impacts N transfer to the host plant in this interaction and that relative N transfer may be possible to predict through basic biochemical analyses.

Tolerability and Acceptability of Autonomous Immersive Virtual Reality Incorporating Digital Twin Technology for Mass Training in Healthcare.

INTRODUCTION: As part of onboarding and systems testing for a clinical expansion, immersive virtual reality (VR) incorporating digital twin technology was used. While digital twin technology has been leveraged by industry, its use in health care has been limited with no prior application for onboarding or training. The tolerability and acceptability of immersive VR for use by a large population of healthcare staff were unknown. METHODS: A prospective, observational study of an autonomous immersive VR onboarding experience to a new clinical space was conducted from May to September 2021. Participants were healthcare staff from several critical care and acute care units. Primary outcomes were tolerance and acceptability measured by reported adverse effects and degree of immersion. Secondary outcomes were attitudes toward the efficacy of VR compared with standard onboarding experiences. RESULTS: A total of 1522 healthcare staff participated. Rates of adverse effects were low and those with prior VR experience were more likely to report no adverse effects. Odds of reporting immersion were high across all demographic groups, though decreased with increasing age. The preference for VR over low-fidelity methods was high across all demographics; however, preferences were mixed when compared with traditional simulation and real-time clinical care. CONCLUSIONS: Large-scale VR onboarding is feasible, tolerable, and acceptable to a diverse population of healthcare staff when using digital twin technology. This study also represents the largest VR onboarding experience to date and may address preconceived notions that VR-based training in health care is not ready for widespread adoption.

Effect of neurosurgical high observation beds (HOBs) on service provision: a single-centre experience and cost analysis.

OBJECTIVE: To evaluate the effect of level 1, high observation beds (HOBs) compared with high dependency unit (HDU) and neurosurgical intensive care unit (NICU) admission on service provision, such as cancelled operations, and healthcare costs. METHODS: A retrospective, observational, single-centre cross-sectional study at a single, large UK neurosurgical centre. All adult patients admitted to neurosurgical HOBs between December 2021 and July 2022 were included. The list of cancelled procedures was collected from 2019 to 2022. To evaluate the impact of admission of eligible patients to HOBs, the total bed days, cost per bed day, number of admissions and cost per admission were obtained for all clinical areas the financial years 2019/2020 and 2021/2022. RESULTS: 307 patients were included in the study: 59.7% of HOBs admissions were elective and 37.7% were acute; admissions were for cranial procedures or conservative treatment (64.8%), spinal (32.6%) or other (2.6%). Following admission, 73.3% of patients were stepped down to the ward prior to discharge home. Only seven patients required escalation to level-2 or level-3 care. Overall, 97% of all HOBs patients were discharged home at the end of hospital stay. Occupancy rate was 90.4%.The cost of bed day increased from ward, level 0 (£384), then level 1 (£376), to level 2 (£787-1211) and to level 3 (£1628). From 2019 to 2021, 558 operations had been cancelled, and 140 (37.8%) of 370 were estimated to have been potentially avoided by HOBs admissions due to conflict of scheduling, ward bed capacity and critical care bed capacity. In addition, a minimum total expenditure due to cancelled operations was estimated at £22 923.50 yearly on average. CONCLUSION: This study recognises HOBs growing role in the management of acutely unwell patients in ward-based environments. While recognising the associated challenges, this study highlighted the potential in reducing healthcare costs. Further studies should evaluate the impact and limitations of HOBs on patients' recovery and outcomes, compared to HDU and NICU.

Cost-effectiveness of repeat delayed imaging for spontaneous subarachnoid hemorrhage.

BACKGROUND: In patients with intracranial aneurysm presenting with spontaneous subarachnoid hemorrhage (SAH), 15% of them could be missed by the initial diagnostic imaging. Repeat delayed imaging can help to identify previously undetected aneurysms, however, the cost-effectiveness of this strategy remains uncertain. OBJECTIVE: The aim of this study is to assess the cost-effectiveness of repeat delayed imaging in patients with SAH who had a negative result during their initial imaging. METHODS: A Markov model was developed to estimate the lifetime costs and quality-adjusted life-year (QALY) for patients who received or not received repeat delayed imaging. The analyses were conducted from a healthcare perspective, with costs reported in UK pounds and expressed in 2020 values. Extensive sensitivity analyses were performed to assess the robustness of the results. RESULTS: The base case incremental cost-effectiveness ratio (ICER) of repeat delayed imaging is £9,314 per QALY compared to no-repeat delayed imaging. This ICER is below the National Institute for Health and Care Excellence (NICE) £20,000 per QALY willingness-to-pay threshold. At the NICE willingness-to-pay threshold of £20,000 per QALY, the probability that repeat delayed imaging is most cost-effective is 0.81. The results are sensitive to age, the utility of survived patients with a favorable outcome, the sensitivity of repeat delayed imaging, and the prevalence of aneurysm. CONCLUSIONS: This study showed that, in the UK, it is cost-effective to provide repeat delayed imaging using computed tomographic angiography (CTA) for patients with SAH who had a negative result in their initial imaging.

Symbiotic status alters fungal eco-evolutionary offspring trajectories.

Despite host-fungal symbiotic interactions being ubiquitous in all ecosystems, understanding how symbiosis has shaped the ecology and evolution of fungal spores that are involved in dispersal and colonization of their hosts has been ignored in life-history studies. We assembled a spore morphology database covering over 26,000 species of free-living to symbiotic fungi of plants, insects and humans and found more than eight orders of variation in spore size. Evolutionary transitions in symbiotic status correlated with shifts in spore size, but the strength of this effect varied widely among phyla. Symbiotic status explained more variation than climatic variables in the current distribution of spore sizes of plant-associated fungi at a global scale while the dispersal potential of their spores is more restricted compared to free-living fungi. Our work advances life-history theory by highlighting how the interaction between symbiosis and offspring morphology shapes the reproductive and dispersal strategies among living forms.

Operational Metrics for the ELAINE 2 Study Combining a Traditional Approach With a Just-in-TIME Model.

PURPOSE: There are numerous barriers to enrollment in oncology biomarker-driven studies. METHODS: The ELAINE 2 study (ClinicalTrials.gov identifier: NCT04432454) is an open-label phase 2 study of lasofoxifene combined with abemaciclib in patients with advanced or metastatic estrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer with an ESR1 mutation. ELAINE 2 opened clinical sites by using a Traditional approach, which activated a site before patient identification, and the Tempus TIME Trial network, which opened a site only after identifying an eligible patient. This manuscript presents the operational metrics comparing the Traditional and TIME Trial site data. RESULTS: The study enrolled patients over 34 weeks and 16 sites (six Traditional and 10 TIME Trial) participated. Duration for full clinical trial agreement execution for Traditional sites and TIME Trial sites averaged 200.5 (range, 142-257) and 7.6 days (range, 2-14), respectively. Institutional review board approval time for Traditional sites and TIME Trial sites was 27.5 (range, 12-71) and 3.0 days (range, 1-12), respectively. Duration from study activation to first consent was 33.3 (range, 18-58) and 8.8 days (range, 1-35) for Traditional and TIME Trial sites, respectively. The first patient on study was at a TIME Trial site 115 days before a Traditional site and the first seven patients enrolled were at TIME Trial sites. Traditional sites consented 23 and enrolled 16 patients, while TIME Trial sites consented 16 and enrolled 13. The trial enrolled 29 patients in 8.5 months with the anticipated enrollment duration being 12-18 months. CONCLUSION: The TIME Trial network opened earlier and enrolled the first study patients. These results demonstrate that the Just-in-TIME model, along with a Traditional model, can improve enrollment in biomarker-driven studies.

Aneurysm management in patients over 80 years old with good grade subarachnoid haemorrhage.

BACKGROUND: An increasing proportion of aneurysmal subarachnoid haemorrhage (aSAH) occurs in older patients, in whom there is widespread variability in treatment rates due to a different balance of risks. Our aim was to compare outcomes of patients over 80 years old with good grade aSAH who underwent treatment of their aneurysm with those who did not. METHODS: Adult patients with good grade aSAH admitted to tertiary regional neurosciences centres contributing to the UK and Ireland Subarachnoid Haemorrhage Database (UKISAH) and a cohort of consecutive patients admitted from three regional cohorts were included for analysis. Outcomes were functional outcome at discharge, three months and survival at discharge. RESULTS: In the UKISAH, patients whose aneurysm was treated were more likely to have a favourable outcome at discharge (OR 2.34, CI 1.12-4.91, p = .02), at three months (OR 2.29, CI 1.11-4.76, p = .04), and lower mortality (10% vs. 29%, OR 0.83, CI 0.72-0.94, p < .01). In the regional cohort, a similar pattern was seen, but after correction for frailty and comorbidity there was no difference in survival (HR 0.45, CI 0.12-1.68, p = .24) or favourable outcome at discharge (OR 0.83, CI 0.23-2.94, p = .77) and at three months (OR 1.03, CI 0.25-4.29, p = .99). CONCLUSIONS: Better early functional outcomes in those undergoing aneurysm treatment appear to be explained by differences in frailty and comorbidity. Therefore, treatment decisions in this patient group are finely balanced with no clear evidence overall of either benefit or harm in this cohort.

Variation along P2RX7 interacts with early traumas on severity of anxiety suggesting a role for neuroinflammation.

Emotional stress is a leading risk factor in the development of neuropsychiatric disorders possibly via immune activation. P2X7 receptors promote neuroinflammation, and research suggests a relationship between chromosome region 12q2431, in which the P2X7R gene is located, and development of mood disorders, however, few studies concentrate on its association with anxiety. Our aim was to investigate the effects of P2RX7 variation in interaction with early childhood traumas and recent stressors on anxiety. 1752 participants completed questionnaires assessing childhood adversities and recent negative life events, provided data on anxiety using the Brief Symptom Inventory, and were genotyped for 681 SNPs in the P2RX7 gene, 335 of which passed quality control and were entered into linear regression models followed by a linkage disequilibrium-based clumping procedure to identify clumps of SNPs with a significant main or interaction effect. We identified a significant clump with top SNP rs67881993 and containing a set of 29SNPs that are in high LD, which significantly interacted with early childhood traumas but not with recent stress conveying a protective effect against increased anxiety in those exposed to early adversities. Our study demonstrated that P2RX7 variants interact with distal and more etiological stressors in influencing the severity of anxiety symptoms, supporting previous scarce results and demonstrating its role in moderating the effects of stress.

Risk of Aneurysm Rupture (ROAR) study: protocol for a long-term, longitudinal, UK multicentre study of unruptured intracranial aneurysms.

INTRODUCTION: Unruptured intracranial aneurysms (UIA) are common in the adult population, but only a relatively small proportion will rupture. It is therefore essential to have accurate estimates of rupture risk to target treatment towards those who stand to benefit and avoid exposing patients to the risks of unnecessary treatment. The best available UIA natural history data are the PHASES study. However, this has never been validated and given the known heterogeneity in the populations, methods and biases of the constituent studies, there is a need to do so. There are also many potential predictors not considered in PHASES that require evaluation, and the estimated rupture risk is largely based on short-term follow-up (mostly 1 year). The aims of this study are to: (1) test the accuracy of PHASES in a UK population, (2) evaluate additional predictors of rupture and (3) assess long-term UIA rupture rates. METHODS AND ANALYSIS: The Risk of Aneurysm Rupture study is a longitudinal multicentre study that will identify patients with known UIA seen in neurosurgery units. Patients will have baseline demographics and aneurysm characteristics collected by their neurosurgery unit and then a single aggregated national cohort will be linked to databases of hospital admissions and deaths to identify all patients who may have subsequently suffered a subarachnoid haemorrhage. All matched admissions and deaths will be checked against medical records to confirm the diagnosis of aneurysmal subarachnoid haemorrhage. The target sample size is 20 000 patients. The primary outcome will be aneurysm rupture resulting in hospital admission or death. Cox regression models will be built to test each of the study's aims. ETHICS AND DISSEMINATION: Ethical approval has been given by South Central Hampshire A Research Ethics Committee (21SC0064) and Confidentiality Advisory Group support (21CAG0033) provided under Section 251 of the NHS Act 2006. The results will be disseminated in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN17658526.

Acquisition of host-derived carbon in biomass of the ectomycorrhizal fungus Pisolithus microcarpus is correlated to fungal carbon demand and plant defences.

Ectomycorrhizal (ECM) fungi are key players in forest carbon (C) sequestration, receiving a substantial proportion of photosynthetic C from their forest tree hosts in exchange for plant growth-limiting soil nutrients. However, it remains unknown whether the fungus or plant controls the quantum of C in this exchange, nor what mechanisms are involved. Here, we aimed to identify physiological and genetic properties of both partners that influence ECM C transfer. Using a microcosm system, stable isotope tracing, and transcriptomics, we quantified plant-to-fungus C transfer between the host plant Eucalyptus grandis and nine isolates of the ECM fungus Pisolithus microcarpus that range in their mycorrhization potential and investigated fungal growth characteristics and plant and fungal genes that correlated with C acquisition. We found that C acquisition by P. microcarpus correlated positively with both fungal biomass production and the expression of a subset of fungal C metabolism genes. In the plant, C transfer was not positively correlated to the number of colonized root tips, but rather to the expression of defence- and stress-related genes. These findings suggest that C acquisition by ECM fungi involves individual fungal demand for C and defence responses of the host against C drain.

Extensive de novo activity stabilizes epigenetic inheritance of CG methylation in Arabidopsis transposons.

Cytosine methylation within CG dinucleotides (mCG) can be epigenetically inherited over many generations. Such inheritance is thought to be mediated by a semiconservative mechanism that produces binary present/absent methylation patterns. However, we show here that, in Arabidopsis thaliana h1ddm1 mutants, intermediate heterochromatic mCG is stably inherited across many generations and is quantitatively associated with transposon expression. We develop a mathematical model that estimates the rates of semiconservative maintenance failure and de novo methylation at each transposon, demonstrating that mCG can be stably inherited at any level via a dynamic balance of these activities. We find that DRM2-the core methyltransferase of the RNA-directed DNA methylation pathway-catalyzes most of the heterochromatic de novo mCG, with de novo rates orders of magnitude higher than previously thought, whereas chromomethylases make smaller contributions. Our results demonstrate that stable epigenetic inheritance of mCG in plant heterochromatin is enabled by extensive de novo methylation.

Speciation Underpinned by Unexpected Molecular Diversity in the Mycorrhizal Fungal Genus Pisolithus.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.