Randomized control trial of a holographic needle guidance technique for thoracic epidural placement.

INTRODUCTION: The Microsoft HoloLens is a head-mounted mixed reality device, which allows for overlaying hologram-like computer-generated elements onto the real world. This technology can be combined with preprocedural ultrasound during thoracic epidural placement to create a visual of the ideal needle angulation and trajectory in the users' field of view. This could result in a technically easier and potentially safer alternative to traditional blind landmark techniques. METHODS: Patients were randomly assigned to one of two groups: (1) HoloLens-assisted thoracic epidural technique (intervention-group H) or (2) traditional thoracic epidural technique (control-group C). The primary outcome was needling time (defined as skin puncture to insertion of epidural catheter) during the procedure. The secondary outcomes were number of needle punctures, number of needle movements, number of bone contacts, and epidural failure. Procedural pain and recovery room pain levels were also evaluated. RESULTS: Eighty-three patients were included in this study. The primary outcome of procedure time was reduced in the HoloLens group compared with control (4.5 min vs 7.3 min, p=0.02, 95% CI), as was the number of needle movements required (7.2 vs 14.4, p=0.01), respectively. There was no difference in intraprocedure or postprocedure pain, bone contacts, or total number of needle punctures. Three patients in the control group experienced epidural failure versus one patient in the HoloLens group. CONCLUSIONS: This study shows that thoracic epidural placement may be facilitated by using a guidance hologram and may be more technically efficient. TRIAL REGISTRATION NUMBER: NCT04028284.

Age and its impact on crisis management performance and learning after simulation-based education by acute care physicians: a multicentre prospective cohort study.

BACKGROUND: Physiological changes associated with ageing could negatively impact the crisis resource management skills of acute care physicians. This study was designed to determine whether physician age impacts crisis resource management skills, and crisis resource management skills learning and retention using full-body manikin simulation training in acute care physicians. METHODS: Acute care physicians at two Canadian universities participated in three 8-min simulated crisis (pulseless electrical activity) scenarios. An initial crisis scenario (pre-test) was followed by debriefing with a trained facilitator and a second crisis scenario (immediate post-test). Participants returned for a third crisis scenario 3-6 months later (retention post-test). RESULTS: For the 48 participants included in the final analysis, age negatively correlated with baseline Global Rating Scale (GRS; r=-0.30, P<0.05) and technical checklist scores (r=-0.44, P<0.01). However, only years in practice and prior simulation experience, but not age, were significant in a subsequent stepwise regression analysis. Learning from simulation-based education was shown with a mean difference in scores from pre-test to immediate post-test of 2.28 for GRS score (P<0.001) and 1.69 for technical checklist correct score (P<0.001); learning was retained for 3-6 months. Only prior simulation experience was significantly correlated with a decreased change in learning (r=-0.30, P<0.05). CONCLUSIONS: A reduced amount of prior simulation training and increased years in practice, but not age on its own, were significant predictors of low baseline crisis resource management performance. Simulation-based education leads to crisis resource management learning that is well retained for 3-6 months, regardless of age or years in practice.

Crosstalk between GABAA receptors in astrocytes and neurons triggered by general anesthetic drugs.

General anesthetic drugs cause cognitive deficits that persist after the drugs have been eliminated. Astrocytes may contribute to such cognition-impairing effects through the release of one or more paracrine factors that increase a tonic inhibitory conductance generated by extrasynaptic γ-aminobutyric acid type A (GABAA) receptors in hippocampal neurons. The mechanisms underlying this astrocyte-to-neuron crosstalk remain unknown. Interestingly, astrocytes express anesthetic-sensitive GABAA receptors. Here, we tested the hypothesis that anesthetic drugs activate astrocytic GABAA receptors to initiate crosstalk leading to a persistent increase in extrasynaptic GABAA receptor function in neurons. We also investigated the signaling pathways in neurons and aimed to identify the paracrine factors released from astrocytes. Astrocytes and neurons from mice were grown in primary cell cultures and studied using in vitro electrophysiological and biochemical assays. We discovered that the commonly used anesthetics etomidate (injectable) and sevoflurane (inhaled) stimulated astrocytic GABAA receptors, which in turn promoted the release paracrine factors, that increased the tonic current in neurons via a p38 MAPK-dependent signaling pathway. The increase in tonic current was mimicked by exogenous IL-1ß and abolished by blocking IL-1 receptors; however, unexpectedly, IL-1ß and other cytokines were not detected in astrocyte-conditioned media. In summary, we have identified a novel form of crosstalk between GABAA receptors in astrocytes and neurons that engages a p38 MAPK-dependent pathway. Brief commentary BACKGROUND: Many older patients experience cognitive deficits after surgery. Anesthetic drugs may be a contributing factor as they cause a sustained increase in the function of "memory blocking" extrasynaptic GABAA receptors in neurons. Interestingly, astrocytes are required for this increase; however, the mechanisms underlying the astrocyte-to-neuron crosstalk remain unknown. TRANSLATIONAL SIGNIFICANCE: We discovered that commonly used general anesthetic drugs stimulate GABAA receptors in astrocytes, which in turn release paracrine factors that trigger a persistent increase in extrasynaptic GABAA receptor function in neurons via p38 MAPK. This novel form of crosstalk may contribute to persistent cognitive deficits after general anesthesia and surgery.

Tranexamic acid for the prevention of postpartum haemorrhage: the TAPPH-1 pilot randomized trial and lessons learned for trials in Canadian obstetrics.

Postpartum haemorrhage (PPH) is a leading cause of maternal morbidity and mortality. While tranexamic acid (TXA) reduces bleeding and transfusion requirements in established PPH, we sought to determine the feasibility of conducting a fully powered trial assessing the effect of prophylactic tranexamic acid, prior to PPH onset, in a Canadian Obstetric setting. With institutional and Health Canada approval, consenting, eligible parturients (singleton, > 32 weeks gestation, vaginal or caesarian delivery) were randomly assigned to receive TXA (1 g intravenously) or placebo (0.9% saline) prior to delivery. Participants, investigators, data collectors/adjudicators, and analysis was blinded. The primary outcome was administration of study intervention to > 85% of randomized individuals. Secondary outcomes included recruitment rate (feasibility) and safety outcomes. Over 8 months, 611 were approached, 35 consented, and 27 randomized (14 TXA, 13 placebo). 89% of randomized participants received the assigned intervention. Recruitment fell below feasibility (23% target). No serious adverse outcomes occurred. Our pilot trial in a Canadian Obstetric setting was unable to demonstrate feasibility to conduct a large, multicentre trial to examine prophylactic use of tranexamic for PPH secondary to the complex regulatory requirements associated with a trial for an off-label, but commonly utilized intervention. These challenges should inform stakeholders on the resources and challenges of conducting future trials using off-label interventions.Trial registration: www.clinicaltrials.gov , NCT03069859 (03/03/2017).

Creating a Successful Virtual Reality-Based Medical Simulation Environment: Tutorial.

Innovation in medical education is not only inevitable but a requirement. Manikin-based simulation is currently the gold standard for supplemental clinical training; however, this modality requires significant equipment and personnel to operate. Virtual reality (VR) is emerging as a new method of delivering medical simulation sessions that requires less infrastructure but also allows for greater accessibility and flexibility. VR has slowly been integrated into the medical curriculum in some hospitals; however, more widespread adoption would transform the delivery of medical education for future clinicians. This tutorial introduces educators to the BUILD REALITY (begin, use, identify, leverage, define, recreate, educate, adapt, look, identify, test, amplify) framework, a series of practical tips for designing and implementing a VR-based medical simulation environment in their curriculum. The suggestions are based on the relevant literature and the authors' personal experience in creating and implementing VR environments for medical trainees. Altogether, this paper provides guidance on conducting a needs assessment, setting objectives, designing a VR environment, and incorporating the session into the broader medical curriculum.

Cognitive deficits after general anaesthesia in animal models: a scoping review.

BACKGROUND: It remains controversial whether general anaesthetic drugs contribute to perioperative neurocognitive disorders in adult patients. Preclinical studies have generated conflicting results, likely because of differing animal models, study protocols, and measured outcomes. This scoping review of preclinical studies addressed the question: 'Do general anaesthetic drugs cause cognitive deficits in adult animals that persist after the drugs have been eliminated from the brain?' METHODS: Reports of preclinical studies in the MEDLINE database published from 1953 to 2021 were examined. A structured review process was used to assess original studies of cognitive behaviours, which were measured after treatment (≥24 h) with commonly used general anaesthetic drugs in adult animals. RESULTS: The initial search yielded 380 articles, of which 106 were fully analysed. The most frequently studied animal model was male (81%; n=86/106) rodents (n=106/106) between 2-3 months or 18-20 months of age. Volatile anaesthetic drugs were more frequently studied than injected drugs, and common outcomes were memory behaviours assessed using the Morris water maze and fear conditioning assays. Cognitive deficits were detected in 77% of studies (n=82/106) and were more frequent in studies of older animals (89%), after inhaled anaesthetics, and longer drug treatments. Limitations of the studies included a lack of physiological monitoring, mortality data, and risk of bias attributable to the absence of randomisation and blinding. CONCLUSIONS: Most studies reported cognitive deficits after general anaesthesia, with age, use of volatile anaesthetic drugs, and duration of anaesthesia as risk factors. Recommendations to improve study design and guide future research are presented.

Perioperative Neurocognitive Screening Tools for At-Risk Surgical Patients.

Purpose of Review: Perioperative neurocognitive disorders are common after surgery and have serious socioeconomic impacts. Despite this, these disorders remain under-recognized and underdiagnosed. To facilitate detection and direct patients toward appropriate preventative interventions, assessment of cognition during the perioperative period is of critical importance. However, there are considerable barriers to the widespread clinical implementation of cognitive assessments, including a lack of consensus regarding the optimal tool for use in specific clinical scenarios. Recent Findings: We provide an overview of the most widely used and validated cognitive assessment tools, including those that permit telemedicine-enabled patient encounters. Summary: No single tool is optimal for all contexts. This narrative review can help clinicians to identify the appropriate cognitive screening tool for their needs by describing the advantages and disadvantages of several available tools, thereby enabling the identification of patients at risk of cognitive decline and facilitating optimization of patient-focused perioperative care.

The Effects of Dexmedetomidine on Perioperative Neurocognitive Outcomes After Cardiac Surgery: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

OBJECTIVE: The purpose of this systematic review and meta-analysis is to examine the effect of DEX on delayed dNCR (cognitive dysfunction ≥ 1 week postoperative) after cardiac surgery. BACKGROUND: DEX has salutary effects on cognitive outcomes following cardiac surgery, however, studies are limited by inconsistent assessment tools, timing, and definitions of dysfunction. It is imperative to identify accurate point estimates of effect of DEX on clinically relevant changes in cognitive function. METHODS: Randomized trials of adults undergoing cardiac surgery comparing perioperative DEX to placebo or alternate sedation and assessing cognitive function ≥ 1 week postoperative were included. Data was abstracted by three reviewers independently and in parallel according to PRISMA guidelines. The primary outcome is dNCR. To classify as dNCR, cognitive function must decrease by at least the minimal clinically important difference or accepted alternate measure (eg, Reliable Change Index ≥1.96). Bias was assessed with the Cochrane Collaboration tool. Data was pooled using a random effects model. RESULTS: Nine trials (942 participants) were included in qualitative analysis, of which seven were included in the meta-analysis of dNCR. DEX reduced the incidence of dNCR (OR 0.39, 95% CI 0.25-0.61, P < 0.0001) compared to placebo/no DEX. There was no difference in the incidence of delirium (OR 0.69, 95% CI 0.35-1.34, P = 0.27) or incidence of hemodynamic instability (OR 1.14, 95% CI 0.59-2.18, P = 0.70) associated with perioperative DEX. CONCLUSIONS: DEX reduced the incidence of dNCR 1 week after cardiac surgery. Although this meta-analysis demonstrates short term cognitive outcomes are improved after cardiac surgery with perioperative DEX, future trials examining long term cognitive outcomes, using robust cognitive assessments, and new perioperative neurocognitive disorders nomenclature with objective diagnostic criteria are necessary.

The Effects of Dexmedetomidine on Perioperative Neurocognitive Outcomes After Noncardiac Surgery: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

Objective: The purpose of this review is to examine the effect of dexmedetomidine on delayed neurocognitive recovery (dNCR; cognitive dysfunction ≥1 week postoperative) after major noncardiac surgery. Background: Dexmedetomidine (DEX) effectively reduces delirium in the intensive care unit and reportedly attenuates cognitive decline following major noncardiac surgery. Ascertaining the true effect on postoperative cognition is difficult because studies are limited by suboptimal selection of cognitive assessment tools, timing of testing, and criteria for defining significant cognitive decline. Methods: Prospective randomized trials comparing perioperative DEX to placebo for major noncardiac surgery assessing cognitive function ≥1 week postoperative were included. Pediatric, nonhuman, and non-English trials, and those where executive function was not assessed were excluded. Data were abstracted by 3 reviewers independently and in parallel according to PRISMA guidelines. The a priori binary primary outcome is dNCR defined as cognitive function declining by the minimal clinically important difference or accepted alternate measure (eg, Reliable Change Index ≥1.96). Bias was assessed with the Cochrane Collaboration tool. Data were pooled using a random effects model. Results: Among 287 citations identified, 26 (9%) met criteria for full-text retrieval. Eleven randomized trials (1233 participants) were included for qualitative analysis, and 7 trials (616 participants) were included for meta-analysis of dNCR. Dexmedetomidine did not reduce the incidence of dNCR significantly (OR 0.57, 95% CI 0.30-1.10, P = 0.09) compared with placebo. There was no difference in the incidence of delirium (OR 0.94, 95% CI 0.55-1.63, P = 0.83) and a higher incidence of hemodynamic instability (OR 2.11, 95% CI 1.22-3.65, P = 0.008). Conclusions: Dexmedetomidine does not reduce dNCR 1 week after major noncardiac surgery. This meta-analysis does not yet support the use of perioperative DEX to improve short term cognitive outcomes at this time; trials underway may yet change this conclusion while larger trials are needed to refine the point estimate of effect and examine long-term cognitive outcomes.

Cognitive Outcomes after DEXmedetomidine sedation in cardiac surgery: CODEX randomised controlled trial protocol.

INTRODUCTION: Older patients undergoing cardiac surgery carry the highest risk for developing major postoperative neurocognitive disorder (postoperative NCD or P-NCD) with up to 25% incidence 3 months after surgery. P-NCD is associated with significant morbidity, mortality, loss of independence, premature retirement and increased healthcare costs. This multicentre randomised trial is investigating the efficacy of postoperative dexmedetomidine sedation in reducing the incidence of major P-NCD after cardiac surgery compared with standard protocols. CODEX will be the largest interventional trial with major P-NCD as the primary outcome. METHODS AND ANALYSIS: CODEX is recruiting patients ≥60 years old, undergoing elective cardiac surgery and without pre-existing major cognitive dysfunction or dementia. Eligible participants are randomised to receive postoperative dexmedetomidine or standard institutional sedation protocols in the intensive care unit. Baseline preoperative cognitive function is assessed with the computer-based Cogstate Brief Battery. The primary outcome, major P-NCD, 3 months after surgery is defined as a decrease in cognitive function ≥1.96 SD below age-matched, non-operative controls. Secondary outcomes include delirium, major P-NCD at 6/12 months, depressive symptoms, mild P-NCD and quality of surgical recovery at 3/6/12 months. The specific diagnostic criteria used in this protocol are consistent with the recommendations for clinical assessment and management of NCD from the Nomenclature Consensus Working Group on perioperative cognitive changes. Intention-to-treat analysis will compare major P-NCD at 3 months between study groups. ETHICS AND DISSEMINATION: CODEX was approved by Sunnybrook Health Sciences Centre Research Ethics Board (REB) (Project ID 1743). This will be the first multicentre, randomised controlled trial to assess the efficacy of a pharmacological intervention to reduce the incidence of major P-NCD after cardiac surgery in patients ≥60 years old. Dissemination of the study results will include briefings of key findings and interpretation, conference presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT04289142.

Cognitive decline among older adults: A hidden preexisting condition and its role in 'brain-at-risk' surgical patients.

Preexisting cognitive impairment is an important, but underrecognized, predictor of postoperative neurocognitive dysfunction, a common and important sequela of surgery. We have applied computerized neuropsychological testing as an efficient and reliable means of detecting preexisting cognitive impairment in two studies of cardiac and noncardiac surgical populations and propose that this tool has great potential in routine clinical diagnosis.

Comparison of the Analgesic Duration of 0.5% Bupivacaine With 1:200,000 Epinephrine Versus 0.5% Ropivacaine Versus 1% Ropivacaine for Low-Volume Ultrasound-Guided Interscalene Brachial Plexus Block: A Randomized Controlled Trial.

BACKGROUND: Bupivacaine and ropivacaine are the preferred long-acting local anesthetics for peripheral nerve blocks as they provide prolonged analgesia in the postoperative period. No studies have directly compared the analgesic duration of these commonly used local anesthetics in the setting of low-volume ultrasound-guided interscalene block (US-ISB). This study was designed to determine which local anesthetic and concentration provides superior analgesia (duration and quality) for low-volume US-ISB. METHODS: Sixty eligible patients scheduled for arthroscopic shoulder surgery were randomized (1:1:1) to receive US-ISB (5 mL) with 0.5% bupivacaine with 1:200,000 epinephrine, 0.5% ropivacaine, or 1% ropivacaine. All individuals were blinded including study participants, anesthesiologists, surgeons, research personnel, and statistician. All participants received a standardized general anesthetic and multimodal analgesia. The primary outcome was duration of analgesia defined as the time from the end of injection to the time that the patients reported a significant increase in pain (>3 numeric rating scale [NRS]) at the surgical site. RESULTS: The mean duration of analgesia for 0.5% bupivacaine with 1:200,000 epinephrine, 0.5% ropivacaine, or 1% ropivacaine was 14.1 ± 7.4, 13.8 ± 4.5, and 15.8 ± 6.3 hours, respectively (analysis of variance [ANOVA], P = .51). There were no observed differences in analgesic duration or other secondary outcomes between the 3 groups with the exception of a difference in cumulative opioid consumption up to 20h00 on the day of surgery in favor of ropivacaine 0.5% over bupivacaine of minimal clinical significance. CONCLUSIONS: In the context of single-injection low-volume US-ISB, we have demonstrated a similar efficacy between equal concentrations of ropivacaine and bupivacaine. In addition, increasing the concentration of ropivacaine from 0.5% to 1% did not prolong the duration of US-ISB.

Pre-hospital hypothermia is associated with transfusion risk after traumatic injury.

OBJECTIVES: In traumatically injured patients, excessive blood loss necessitating the transfusion of red blood cell (RBC) units is common. Indicators of early RBC transfusion in the pre-hospital setting are needed. This study aims to evaluate the association between hypothermia (<36°C) and transfusion risk within the first 24 hours after arrival to hospital for a traumatic injury. METHODS: We completed an audit of all traumatically injured patients who had emergent surgery at a single tertiary care center between 2010 and 2014. Using multivariable logistic regression analysis, we evaluated the association between pre-hospital hypothermia and transfusion of ≥1 unit of RBC within 24 hours of arrival to the trauma bay. RESULTS: Of the 703 patients included to evaluate the association between hypothermia and RBC transfusion, 203 patients (29%) required a transfusion within 24 hours. After controlling for important confounding variables, including age, sex, coagulopathy (platelets and INR), hemoglobin, and vital signs (blood pressure and heart rate), hypothermia was associated with a 68% increased odds of transfusion in multivariable analysis (OR: 1.68; 95% CI: 1.11-2.56). CONCLUSIONS: Hypothermia is strongly associated with RBC transfusion in a cohort of trauma patients requiring emergent surgery. This finding highlights the importance of early measures of temperature after traumatic injury and the need for intervention trials to determine if strategies to mitigate the risk of hypothermia will decrease the risk of transfusion and other morbidities.

Comparison of the effects of perineural or intravenous dexamethasone on low volume interscalene brachial plexus block: a randomised equivalence trial.

BACKGROUND: Efforts to prolong interscalene block (ISB) analgesia include the use of local anaesthetic adjuvants such as dexamethasone. Previous work showing prolonged block duration suggests that both perineural and intravenous (i.v.) routes can both prolong analgesia. The superiority of either route is controversial given the design of previous studies. As perineural dexamethasone is an off-label use, anaesthesiologists should be fully informed of the clinical differences, if any, on block duration. This study was designed to test whether perineural vs i.v. dexamethasone administration are equivalent. METHODS: We randomised 182 eligible patients scheduled for arthroscopic shoulder surgery to receive low-dose ISB (0.5% ropivacaine 5 ml) with perineural or i.v. dexamethasone 4 mg. Subjects, anaesthesiologists, and research personnel were blinded. All subjects also received a standardised general anaesthetic and multimodal analgesia. The primary outcome was duration of analgesia analysed as an equivalence outcome (2 h equivalency margin) using the two one-sided test (TOST) method. RESULTS: For the primary outcome, duration of analgesia, and perineural and i.v. administration of dexamethasone were not equivalent. The upper and lower bounds of the 90% confidence interval were 1 h (P=0.12) and -2.5 h (P=0.01), respectively. The observed difference in mean block duration was not clinically relevant (0.75 h longer for i.v. dexamethasone). There were no other clinically significant differences between groups. CONCLUSION: In the context of low-volume ISB with ropivacaine, perineural and i.v. dexamethasone were not equivalent in terms of their effects on block duration. However, there were no clinically significant differences in outcomes, and there is no advantage of perineural over intravenous dexamethasone. WWW.CLINICALTRIALS. GOV REGISTRATION: NCT02322242.

The MLL1 trimeric catalytic complex is a dynamic conformational ensemble stabilized by multiple weak interactions.

Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. The catalytic activity of this family is dependent on interactions with additional conserved proteins, but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering, cross-linking mass spectrometry, nuclear magnetic resonance spectroscopy and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 ß-propeller domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

Conformational dynamics of the TTD-PHD histone reader module of the UHRF1 epigenetic regulator reveals multiple histone-binding states, allosteric regulation, and druggability.

UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy. Recent studies indicate that interdomain interactions critically influence UHRF1's chromatin-binding properties, including allosteric regulation of its histone binding. Here, using an integrative approach that combines small angle X-ray scattering, NMR spectroscopy, and molecular dynamics simulations, we characterized the dynamics of the tandem tudor domain-plant homeodomain (TTD-PHD) histone reader module, including its 20-residue interdomain linker. We found that the apo TTD-PHD module in solution comprises a dynamic ensemble of conformers, approximately half of which are compact conformations, with the linker lying in the TTD peptide-binding groove. These compact conformations are amenable to cooperative, high-affinity histone binding. In the remaining conformations, the linker position was in flux, and the reader adopted both extended and compact states. Using a small-molecule fragment screening approach, we identified a compound, 4-benzylpiperidine-1-carboximidamide, that binds to the TTD groove, competes with linker binding, and promotes open TTD-PHD conformations that are less efficient at H3K9me3 binding. Our work reveals a mechanism by which the dynamic TTD-PHD module can be allosterically targeted with small molecules to modulate its histone reader function for therapeutic or experimental purposes.

UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119.

UbE2E1/UbcH6 is an E2 ubiquitin-conjugating enzyme that is regulated by USP7. We identified UbE2E1 as a novel component of Polycomb repressive complex 1 (PRC1), the E3 ligase complex responsible for histone H2A ubiquitination and gene silencing. We demonstrate that UbE2E1 is critical for the monoubiquitination of H2A at residue Lys-119 (uH2AK119) through its association with the PRC1 complex. UbE2E1 interacts with PRC1 subunits including Ring1A and Ring1B. Overexpression of UbE2E1 results in increased levels of uH2AK119, whereas overexpression of catalytically inactive UbE2E1_C131A or UbE2E1 knockdown results in decreased levels of uH2AK119. The down-regulation of H2A ubiquitination by loss of function of UbE2E1 is correlated with alleviated p16INK4a promoter repression and induced growth inhibition in HCT116 cells. These results are specific to UbE2E1 as knockdown of UbE2D E2s does not show any effect on uH2AK119. We extended the UbE2E1 regulation of uH2AK119 to USP7 and showed that USP7 is also a key regulator for monoubiquitination at H2A Lys-119 as both knockdown and deletion of USP7 results in decreased levels of uH2AK119. This study reveals that UbE2E1 is an in vivo E2 for the PRC1 ligase complex and thus plays an important role in the regulation of H2A Lys-119 monoubiquitination.

Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.

Cbx2 targets PRC1 to constitutive heterochromatin in mouse zygotes in a parent-of-origin-dependent manner.

Polycomb repressive complexes PRC1 and PRC2 regulate expression of genes involved in proliferation and development. In mouse early embryos, however, canonical PRC1 localizes to paternal pericentric heterochromatin (pat-PCH), where it represses transcription of major satellite repeats. In contrast, maternal PCH (mat-PCH) is enriched for H3 lysine 9 tri-methylation (H3K9me3) and Hp1ß. How PRC1 is targeted to pat-PCH, yet excluded from mat-PCH, has remained elusive. Here, we identify a PRC1 targeting mechanism that relies on Cbx2 and Hp1ß. Cbx2 directs catalytically active PRC1 to PCH via its chromodomain (CD(Cbx2)) and neighboring AT-hook (AT(Cbx2)) binding to H3K27me3 and AT-rich major satellites, respectively. CD(Cbx2) prevents AT(Cbx2) from interacting with DNA at PCH marked by H3K9me3 and Hp1ß. Loss-of-function studies show that Hp1ß and not H3K9me3 prevents PRC1 targeting to mat-PCH. Our findings indicate that CD(Cbx2) and AT(Cbx2) separated by a short linker function together to integrate H3K9me3/HP1 and H3K27me3 states.

KCMF1 (potassium channel modulatory factor 1) Links RAD6 to UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4) and lysosome-mediated degradation.

RAD6 is a ubiquitin E2 protein with roles in a number of different biological processes. Here, using affinity purification coupled with mass spectrometry, we identify a number of new RAD6 binding partners, including the poorly characterized ubiquitin E3 ligases KCMF1 (potassium channel modulatory factor 1) and UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4), a protein that can bind N-end rule substrates, and which was recently linked to lysosome-mediated degradation and autophagy. NMR, combined with in vivo and in vitro interaction mapping, demonstrate that the KCMF1 C terminus binds directly to RAD6, whereas N-terminal domains interact with UBR4 and other intracellular vesicle- and mitochondria-associated proteins. KCMF1 and RAD6 colocalize at late endosomes and lysosomes, and cells disrupted for KCMF1 or RAD6 function display defects in late endosome vesicle dynamics. Notably, we also find that two different RAD6A point mutants (R7W and R11Q) found in X-linked intellectual disability (XLID) patients specifically lose the interaction with KCMF1 and UBR4, but not with other previously identified RAD6 interactors. We propose that RAD6-KCMF1-UBR4 represents a unique new E2-E3 complex that targets unknown N-end rule substrates for lysosome-mediated degradation, and that disruption of this complex via RAD6A mutations could negatively affect neuronal function in XLID patients.