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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. [ABSTRACT FROM AUTHOR]

The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1-8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity. [ABSTRACT FROM AUTHOR]

Cul7 is a member of the Cullin Ring Ligase (CRL) family and is required for normal mouse development and cellular proliferation. Recently, a region of Cul7 that is highly conserved in the p53-associated, Parkin-like cytoplasmic protein PARC, was shown to bind p53 directly. Here we identify the CPH domains (conserved domain within Cul7, PARC, and HERC2 proteins) of both Cul7 and PARC as p53 interaction domains using size exclusion chromatography and NMR spectroscopy. We present the first structure of the evolutionarily conserved CPH domain and provide novel insight into the Cul7-p53 interaction. The NMR structure of the Cul7-CPH domain reveals a fold similar to peptide interaction modules such as the SH3, Tudor, and KOW domains. The p53 interaction surface of both Cul7 and PARC CPH domains was mapped to a conserved surface distinct from the analogous peptide-binding regions of SH3, KOW, and Tudor domains, suggesting a novel mode of interaction. The CPH domain interaction surface of p53 resides in the tetramerization domain and is formed by residues contributed by at least two subunits. [ABSTRACT FROM AUTHOR]

One of many protein-protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1-120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37-57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37-57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic pS3N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage. [ABSTRACT FROM AUTHOR]

Proton decoupled 31P and 13C solution NMR experiments were applied to mixtures of 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase, with each of its natural substrates, phosphoenolpyruvate and arabinose-5-phosphate (ASP), and product KDO8P to identify the formation of the enzyme–substrate and enzyme–product complexes. Effects arising from ligand interactions with the enzyme are reported via chemical shifts and line broadening with respect to those of the free ligands in solution, depending on the strength and dynamics of binding under thermodynamic equilibrium conditions. The characterization was done both at low and high field spectrometers, 200 and 500 MHz (1H frequencies), and in cases of 31P NMR measurements, it was demonstrated that only the low field spectrometer is capable of providing direct experimental evidence on the enzyme–ligand interactions. Since both the substrate A5P and the product KDO8P exhibit multiple anomeric forms in solution, evidence for the preference of recognition and binding of particular forms is sought. [Copyright &y& Elsevier]

In this report the mode of inhibition of mechanism-based inhibitor (2, K[sub i] = 0.4 µM) of 3-deoxyD-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which was designed to mimic the combined key features of its natural substrates arabinose-5-phosphate (A5P) and phoshoenolpyruvate (PEP) into a single molecule, was investigated. Our earlier solid-state NMR observations identified the inhibitor to bind in a way that partly mimics ASP, while the phosphonate moiety of its PEP-mimicking part exhibits no interactions with enzyme residues. This result was apparently in disagreement with the competitive inhibition of 2 against PEP and with the later solved crystal structure of KDO8PS-2 binary complex identifying the interactions of its PEP-mimicking part with the enzyme residues that were not detected by solid-state NMR. To solve this discrepancy, further solid-state REDOR NMR and [sup 31]P solution NMR experiments were applied to a variety of enzyme complexes with the substrates and inhibitor. In particular, a novel frequency-selective REDOR experiment was developed and applied. Integration of the solution and solid-state NMR data clearly demonstrates that under conditions of stoichiometric enzyme-ligand ratio at thermodynamic equilibrium (a) PEP binding is unperturbed by the presence of 2 and (b) both PEP and 2 can bind simultaneously to the synthase, i.e., form a ternary complex with PEP occupying its own subsite and 2 occupying A5P's subsite. The latter observation suggests that under the conditions used in our NMR measurements, the inhibition pattern of 2 against PEP should have a mixed type character. Furthermore, the NMR data directly demonstrate the distinction between the relative binding strength of the two moieties of 2: enzyme interactions with PEP-mimicking moiety are much weaker than those with the A5P moiety. This observation is in agreement with KDO8PS-2 crystal structure showing only remote contacts of the phosphonate due to large structural... [ABSTRACT FROM AUTHOR]

Investigates the mechanism of action of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase. Use of solid-state REDOR nuclear magnetic resonance; Types of enzyme residues that bind the phosphate groups of each ligand; Structure of each basic enzyme residue.

Hip replacement surgery can be painful; postoperative analgesia is crucial for comfort and to facilitate recovery. Regional anaesthesia can reduce pain and postoperative opioid requirements. The role of ultrasound-guided suprainguinal fascia iliaca block for analgesia after elective total hip arthroplasty is not well defined. This randomised trial evaluated its analgesic efficacy. Consenting participants (134) scheduled for elective primary total hip arthroplasty under spinal anaesthesia were randomly allocated to receive ultrasound-guided fascia iliaca block with ropivacaine 0.5% or sham block with saline. The primary outcome was opioid consumption in the first 24 h after surgery. Additional outcomes included pain scores at 4, 8, 12, and 16 h, opioid-related side-effects (nausea, vomiting, pruritis), ability to perform physiotherapy on the first postoperative day, and physiotherapist-assessed quadriceps weakness. There were no significant differences in 24-h opioid consumption (block vs sham block, mean difference −3.2 mg oral morphine equivalent, 95% confidence interval −15.3 to 8.1 mg oral morphine equivalent, P =0.55) or any other prespecified outcomes. In patients undergoing primary total hip arthroplasty, ultrasound-guided suprainguinal fascia iliaca block with ropivacaine did not confer a significant opioid-sparing effect compared with sham block. There were no differences in other secondary outcomes including pain scores, opioid-related side-effects, or ability to perform physiotherapy on the first postoperative day. www.clinicaltrials.gov (NCT03069183). [ABSTRACT FROM AUTHOR]

Presents a study of spin transition in a manganese(III) porphyrin cation radical, its transformation to a dichloromanganese(IV) porphyrin, and chlorination of hydrocarbons by the latter. Effect of chemical oxidation of (TMP)MnIII(CI) (TMP=the tetramesitylporphyrinato dianion by Fe(CIO4); Detailed information on study.

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. 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). 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). 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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Approximately 31% of cardiac surgery patients who completed baseline CBB testing had major pre-CI (20 of 64 patients have already been tested, from a projected sample size of 2,400). Another study of 215 older adults who were undergoing major elective surgery, similarly reported that 121 patients had performance criteria of pre-CI, yet only two patients had any prior documented diagnosis of cognitive impairment (Smith & Yeow, 2016). Keywords: anesthesia; cognitive assessment screening instrument; mild neurocognitive disorder; neurocognitive tests; postoperative cognitive disorder EN anesthesia cognitive assessment screening instrument mild neurocognitive disorder neurocognitive tests postoperative cognitive disorder 1 6 6 05/19/21 20210501 NES 210501 Preexisting cognitive impairment is an important, but underrecognized, predictor of postoperative neurocognitive dysfunction, a common and important sequela of surgery. While P-NCD can affect any patient after surgery, its incidence and consequences are highest among older adults with rates estimated to be between 10% and 30% at 3 months after surgery, depending on surgical procedure (Knipp et al., 2017; Moller et al., 1998; Monk et al., 2008). [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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 smallmolecule 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 atH3K9me3 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary 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 Hp1β. 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 Hp1β. 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 Hp1β. Loss-of-function studies show that Hp1β 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. [ABSTRACT FROM AUTHOR]

Summary: The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response. [Copyright &y& Elsevier]

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2-/- mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor. [ABSTRACT FROM AUTHOR]