Global identification of SWI/SNF targets reveals compensation by EP400.

Mammalian SWI/SNF chromatin remodeling complexes move and evict nucleosomes at gene promoters and enhancers to modulate DNA access. Although SWI/SNF subunits are commonly mutated in disease, therapeutic options are limited by our inability to predict SWI/SNF gene targets and conflicting studies on functional significance. Here, we leverage a fast-acting inhibitor of SWI/SNF remodeling to elucidate direct targets and effects of SWI/SNF. Blocking SWI/SNF activity causes a rapid and global loss of chromatin accessibility and transcription. Whereas repression persists at most enhancers, we uncover a compensatory role for the EP400/TIP60 remodeler, which reestablishes accessibility at most promoters during prolonged loss of SWI/SNF. Indeed, we observe synthetic lethality between EP400 and SWI/SNF in cancer cell lines and human cancer patient data. Our data define a set of molecular genomic features that accurately predict gene sensitivity to SWI/SNF inhibition in diverse cancer cell lines, thereby improving the therapeutic potential of SWI/SNF inhibitors.

Unique and Shared Epigenetic Programs of the CREBBP and EP300 Acetyltransferases in Germinal Center B Cells Reveal Targetable Dependencies in Lymphoma.

Inactivating mutations of the CREBBP and EP300 acetyltransferases are among the most common genetic alterations in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). Here, we examined the relationship between these two enzymes in germinal center (GC) B cells, the normal counterpart of FL and DLBCL, and in lymphomagenesis by using conditional GC-directed deletion mouse models targeting Crebbp or Ep300. We found that CREBBP and EP300 modulate common as well as distinct transcriptional programs implicated in separate anatomic and functional GC compartments. Consistently, deletion of Ep300 but not Crebbp impaired the fitness of GC B cells in vivo. Combined loss of Crebbp and Ep300 completely abrogated GC formation, suggesting that these proteins partially compensate for each other through common transcriptional targets. This synthetic lethal interaction was retained in CREBBP-mutant DLBCL cells and could be pharmacologically targeted with selective small molecule inhibitors of CREBBP and EP300 function. These data provide proof-of-principle for the clinical development of EP300-specific inhibitors in FL and DLBCL.

Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding.

Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.

p300-Mediated Lysine 2-Hydroxyisobutyrylation Regulates Glycolysis.

Lysine 2-hydroxyisobutyrylation (Khib) is an evolutionarily conserved and widespread histone mark like lysine acetylation (Kac). Here we report that p300 functions as a lysine 2-hyroxyisobutyryltransferase to regulate glycolysis in response to nutritional cues. We discovered that p300 differentially regulates Khib and Kac on distinct lysine sites, with only 6 of the 149 p300-targeted Khib sites overlapping with the 693 p300-targeted Kac sites. We demonstrate that diverse cellular proteins, particularly glycolytic enzymes, are targeted by p300 for Khib, but not for Kac. Specifically, deletion of p300 significantly reduces Khib levels on several p300-dependent, Khib-specific sites on key glycolytic enzymes including ENO1, decreasing their catalytic activities. Consequently, p300-deficient cells have impaired glycolysis and are hypersensitive to glucose-depletion-induced cell death. Our study reveals an p300-catalyzed, Khib-specific molecular mechanism that regulates cellular glucose metabolism and further indicate that p300 has an intrinsic ability to select short-chain acyl-CoA-dependent protein substrates.

Prostaglandin E2 activates melanin-concentrating hormone neurons to drive diet-induced obesity.

Hypothalamic inflammation reduces appetite and body weight during inflammatory diseases, while promoting weight gain when induced by high-fat diet (HFD). How hypothalamic inflammation can induce opposite energy balance outcomes remains unclear. We found that prostaglandin E2 (PGE2), a key hypothalamic inflammatory mediator of sickness, also mediates diet-induced obesity (DIO) by activating appetite-promoting melanin-concentrating hormone (MCH) neurons in the hypothalamus in rats and mice. The effect of PGE2 on MCH neurons is excitatory at low concentrations while inhibitory at high concentrations, indicating that these neurons can bidirectionally respond to varying levels of inflammation. During prolonged HFD, endogenous PGE2 depolarizes MCH neurons through an EP2 receptor-mediated inhibition of the electrogenic Na+/K+-ATPase. Disrupting this mechanism by genetic deletion of EP2 receptors on MCH neurons is protective against DIO and liver steatosis in male and female mice. Thus, an inflammatory mediator can directly stimulate appetite-promoting neurons to exacerbate DIO and fatty liver.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.

A UTX-MLL4-p300 Transcriptional Regulatory Network Coordinately Shapes Active Enhancer Landscapes for Eliciting Transcription.

Enhancer activation is a critical step for gene activation. Here we report an epigenetic crosstalk at enhancers between the UTX (H3K27 demethylase)-MLL4 (H3K4 methyltransferase) complex and the histone acetyltransferase p300. We demonstrate that UTX, in a demethylase activity-independent manner, facilitates conversion of inactive enhancers in embryonic stem cells to an active (H3K4me1+/H3K27ac+) state by recruiting and coupling the enzymatic functions of MLL4 and p300. Loss of UTX leads to attenuated enhancer activity, characterized by reduced levels of H3K4me1 and H3K27ac as well as impaired transcription. The UTX-MLL4 complex enhances p300-dependent H3K27 acetylation through UTX-dependent stimulation of p300 recruitment, while MLL4-mediated H3K4 monomethylation, reciprocally, requires p300 function. Importantly, MLL4-generated H3K4me1 further enhances p300-dependent transcription. This work reveals a previously unrecognized cooperativity among enhancer-associated chromatin modulators, including a unique function for UTX, in establishing an "active enhancer landscape" and defines a detailed mechanism for the joint deposition of H3K4me1 and H3K27ac.

EP300/CREBBP acetyltransferase inhibition limits steroid receptor and FOXA1 signaling in prostate cancer cells.

The androgen receptor (AR) is a primary target for treating prostate cancer (PCa), forming the bedrock of its clinical management. Despite their efficacy, resistance often hampers AR-targeted therapies, necessitating new strategies against therapy-resistant PCa. These resistances involve various mechanisms, including AR splice variant overexpression and altered activities of transcription factors like the glucocorticoid receptor (GR) and FOXA1. These factors rely on common coregulators, such as EP300/CREBBP, suggesting a rationale for coregulator-targeted therapies. Our study explores EP300/CREBBP acetyltransferase inhibition's impact on steroid receptor and FOXA1 signaling in PCa cells using genome-wide techniques. Results reveal that EP300/CREBBP inhibition significantly disrupts the AR-regulated transcriptome and receptor chromatin binding by reducing the AR-gene expression. Similarly, GR's regulated transcriptome and receptor binding were hindered, not linked to reduced GR expression but to diminished FOXA1 chromatin binding, restricting GR signaling. Overall, our findings highlight how EP300/CREBBP inhibition distinctively curtails oncogenic transcription factors' signaling, suggesting the potential of coregulatory-targeted therapies in PCa.

cAMP competitively inhibits periplasmic phosphatases to coordinate nutritional growth with competence of Haemophilus influenzae.

Most naturally competent bacteria tightly regulate the window of the competent state to maximize their ecological fitness under specific conditions. Development of competence by Haemophilus influenzae strain Rd KW20 is stimulated by cAMP and inhibited by purine nucleotides, respectively. In contrast, cAMP inhibits cell growth, but nucleotides are important for KW20 growth. However, the mechanisms underlying the abovementioned reciprocal effects are unclear. Here, we first identified a periplasmic acid phosphatase AphAEc of Escherichia coli as a new cAMP-binding protein. We show cAMP competitively inhibits the phosphatase activities of AphAEc and its homolog protein AphAHi in the KW20 strain. Furthermore, we found cAMP inhibits two other periplasmic nonspecific phosphatases, NadNHi (which provides the essential growth factor V, NAD) and HelHi (eP4, which converts NADP to NAD) in KW20. We demonstrate cAMP inhibits cell growth rate, especially via NadNHi. On the other hand, the inhibitory effect of purine nucleotide AMP on competence was abolished in the triple deletion mutant ΔhelHiΔnadNHiΔaphAHi, but not in the single, double deletion or complemented strains. Adenosine, however, still inhibited the competence of the triple deletion mutant, demonstrating the crucial role of the three phosphatases in converting nucleotides to nucleosides and thus inhibiting KW20 competence. Finally, cAMP restored the competence inhibited by GMP in a dose-dependent manner, but not competence inhibited by guanosine. Altogether, we uncovered these three periplasmic phosphatases as the key players underlying the antagonistic effects of cAMP and purine nucleotides on both cell growth and competence development of H. influenzae.

Salmonella engages CDC42 effector protein 1 for intracellular invasion.

Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread.

Inducible deletion of the prostaglandin EP3 receptor in kidney tubules of male and female mice has no major effect on water homeostasis.

The prostaglandin E2 (PGE2) receptor EP3 has been detected in the thick ascending limb (TAL) and the collecting duct of the kidney, where its actions are proposed to inhibit water reabsorption. However, EP3 is also expressed in other cell types, including vascular endothelial cells. The aim here was to determine the contribution of EP3 in renal water handling in male and female adult mice by phenotyping a novel mouse model with doxycycline-dependent deletion of EP3 throughout the kidney tubule (EP3-/- mice). RNAscope demonstrated that EP3 was highly expressed in the cortical and medullary TAL of adult mice. Compared with controls EP3 mRNA expression was reduced by >80% in whole kidney (RT-qPCR) and nondetectable (RNAscope) in renal tubules of EP3-/- mice. Under basal conditions, there were no significant differences in control and EP3-/- mice of both sexes in food and water intake, body weight, urinary output, or clinical biochemistries. No differences were detectable between genotypes in handling of an acute water load or in their response to the vasopressin analog 1-deamino-8-d-arginine-vasopressin (dDAVP). No differences in water handling were observed when PGE2 production was enhanced using 1% NaCl load. Expression of proteins involved in kidney water handling was not different between genotypes. This study demonstrates that renal tubular EP3 is not essential for body fluid homeostasis in males or females, even when PGE2 levels are high. The mouse model is a novel tool for examining the role of EP3 in kidney function independently of potential developmental abnormalities or systemic effects.NEW & NOTEWORTHY The prostanoid EP3 receptor is proposed to play a key role in the kidney tubule and antagonize the effects of vasopressin on aquaporin-mediated water reabsorption. Here, we phenotyped a kidney tubule-specific inducible knockout mouse model of the EP3 receptor. Our major finding is that, even under physiological stress, tubular EP3 plays no detectable role in renal water or solute handling. This suggests that other EP receptors must be important for renal salt and water handling.

EP300 promotes lung cancer cell proliferation by regulating the oncogenic transcription of Hippo-YAP signaling pathway.

The transcriptional activation function of YAP in cancer development has been widely studied. However, the underlying regulatory mechanisms remain largely unknown. In this study, we found that EP300, one histone acetyltransferase, interacted with YAP and was recruited into the phase separated condensates of YAP. Transcriptomic analysis revealed substantial alterations in gene expression upon EP300 depletion, with downregulated genes associated with cancer progression and Hippo-YAP pathway. Notably, disruption of EP300 inhibited the transcriptional activation of YAP and reduced the binding of H3K27ac on YAP target oncogenes in Hippo pathway. Moreover, depletion of EP300 effectively inhibited YAP-driven tumor growth. Taken together, these results indicate that EP300 contributes to lung cancer progression by promoting the oncogenic transcription of YAP through H3K27ac, which suggests that YAP-EP300 axis may be potential therapeutic targets for lung cancer treatment.

NDUFA8 is transcriptionally regulated by EP300/H3K27ac and promotes mitochondrial respiration to support proliferation and inhibit apoptosis in cervical cancer.

Cervical cancer, a common malignancy in women, poses a significant health burden worldwide. In this study, we aimed to investigate the expression, function, and potential mechanisms of NADH: ubiquinone oxidoreductase subunit A8 (NDUFA8) in cervical cancer. The Gene Expression Profiling Interactive Analysis (GEPIA) database and immunohistochemical scoring were used to analyze NDUFA8 expression in cervical cancer tissues and normal tissues. Quantitative real-time PCR and Western blot analyses were performed to assess the expression level of NDUFA8 in cervical cancer cell lines. NDUFA8 knockdown or overexpression experiments were conducted to evaluate its impact on cell proliferation and apoptosis. The mitochondrial respiratory status was analyzed by measuring cellular oxygen consumption, adenosine triphosphate (ATP) levels, and the expression levels of Mitochondrial Complex I activity, and Mitochondrial Complex IV-associated proteins Cytochrome C Oxidase Subunit 5B (COX5B) and COX6C. NDUFA8 exhibited high expression levels in cervical cancer tissues, and these levels were correlated with reduced survival rates. A significant upregulation of NDUFA8 expression was observed in cervical cancer cell lines compared to normal cells. Silencing NDUFA8 hindered cell proliferation, promoted apoptosis, and concurrently suppressed cellular mitochondrial respiration, resulting in decreased levels of available ATP. Conversely, NDUFA8 overexpression induced the opposite effects. Herein, we also found that E1A Binding Protein P300 (EP300) overexpression facilitated Histone H3 Lysine 27 (H3K27) acetylation enrichment, enhancing the activity of the NDUFA8 promoter region. NDUFA8, which is highly expressed in cervical cancer, is regulated by transcriptional control via EP300/H3K27 acetylation. By promoting mitochondrial respiration, NDUFA8 contributes to cervical cancer cell proliferation and apoptosis. These findings provide novel insights into NDUFA8 as a therapeutic target in cervical cancer.

Berberine alleviates inflammation and suppresses PLA2-COX-2-PGE2-EP2 pathway through targeting gut microbiota in DSS-induced ulcerative colitis.

Berberine, isolated from Coptis chinensis and Phellodendron amurense, can attenuate colonic injury and modulate gut microbiota disorders in ulcerative colitis (UC). However, the mechanism and causal relationship between gut microbiota and the efficacy of Berberine on UC are still unclear, which were investigated by pseudo-germ-free (PGF) mice, 16S rRNA gene analysis and transcriptome analysis in this study. The results demonstrated that Berberine improved gut microbiota disorders, colon damage, tight-junction proteins, inflammatory and anti-inflammatory cytokines in DSS-induced colitis mice with intact gut microbiota but not in PGF mice. Besides, immune-related and inflammation-related pathways were closely related to the efficacy that Berberine alleviated colitis by regulating gut microbiota. Furthermore, Berberine reduced PGE2, PLA2, COX-2, Ptges, EP2 and p-Stat3 only in colitis mice with intact gut microbiota. In summary, our study confirms that Berberine inhibits PLA2-COX-2-PGE2-EP2 pathway in UC through gut microbiota, leading to the alleviation of inflammation in colon, which further elucidates the underlying mechanism and promotes the application of Berberine in UC.

Posttranscriptional regulation of the prostaglandin E receptor spliced-isoform EP3-γ and its implication in pancreatic ß-cell failure.

In Type 2 diabetes (T2D), elevated lipid levels have been suggested to contribute to insulin resistance and ß-cell dysfunction. We previously reported that the expression of the PGE2 receptor EP3 is elevated in islets of T2D individuals and is preferentially stimulated by palmitate, leading to ß-cell failure. The mouse EP3 receptor generates three isoforms by alternative splicing which differ in their C-terminal domain and are referred to as mEP3α, mEP3ß, and mEP3γ. We bring evidence that the expression of the mEP3γ isoform is elevated in islets of diabetic db/db mice and is selectively upregulated by palmitate. Specific knockdown of the mEP3γ isoform restores the expression of ß-cell-specific genes and rescues MIN6 cells from palmitate-induced dysfunction and apoptosis. This study indicates that palmitate stimulates the expression of the mEP3γ by a posttranscriptional mechanism, compared to the other spliced isoforms, and that the de novo synthesized ceramide plays an important role in FFA-induced mEP3γ expression in ß-cells. Moreover, induced levels of mEP3γ mRNA by palmitate or ceramide depend on p38 MAPK activation. Our findings suggest that mEP3γ gene expression is regulated at the posttranscriptional level and defines the EP3 signaling axis as an important pathway mediating ß-cell-impaired function and demise.

Rare case of simultaneous occurrence of chronic neutrophil leukemia and T lymphoblastic lymphoma: case report and literature review.

Chronic neutrophil leukemia (CNL) is a rare and life-threatening disease. Cases of CNL combined with lymphoma are rare. Here, we report a case of CNL with T-acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) in a 28-year-old male. After a regimen of ruxolitinib, VICLP (Vincristine, Idarubicin, Cyclophosphamide, Prednisone, Peg-asparaginase) regimen, high-dose cytarabine, and methotrexate regimens, the patient's bone marrow condition partially resolved. However, when the disease relapsed four months later, despite attempts with selinexor, venetoclax, and CAG(aclarubicin hydrochloride, Algocytidine, Granulocyte Stimulating Factor) chemotherapy, the leukocytes and peripheral blood primitive cells reduced, but the bone marrow did not achieve remission. This pathogenesis may be related to microenvironmental immune escape under prolonged inflammatory stimulation and gene disruption affecting protein function due to colony-stimulating factor 3 receptor gene (CSF3R) mutations. For this type of disease, early intervention may delay disease progression.

EP300-ZNF384 transactivates IL3RA to promote the progression of B-cell acute lymphoblastic leukemia.

The EP300-ZNF384 fusion gene is an oncogenic driver in B-cell acute lymphoblastic leukemia (B-ALL). In the present study, we demonstrated that EP300-ZNF384 substantially induces the transcription of IL3RA and the expression of IL3Rα (CD123) on B-ALL cell membranes. Interleukin 3 (IL-3) supplementation promotes the proliferation of EP300-ZNF348-positive B-ALL cells by activating STAT5. Conditional knockdown of IL3RA in EP300-ZF384-positive cells inhibited the proliferation in vitro, and induced a significant increase in overall survival of mice, which is attributed to impaired propagation ability of leukemia cells. Mechanistically, the EP300-ZNF384 fusion protein transactivates the promoter activity of IL3RA by binding to an A-rich sequence localized at -222/-234 of IL3RA. Furthermore, forced EP300-ZNF384 expression induces the expression of IL3Rα on cell membranes and the secretion of IL-3 in CD19-positive B precursor cells derived from healthy individuals. Doxorubicin displayed a selective killing of EP300-ZNF384-positive B-ALL cells in vitro and in vivo. Collectively, we identify IL3RA as a direct downstream target of EP300-ZNF384, suggesting CD123 is a potent biomarker for EP300-ZNF384-driven B-ALL. Targeting CD123 may be a novel therapeutic approach to EP300-ZNF384-positive patients, alternative or, more likely, complementary to standard chemotherapy regimen in clinical setting.

Utilization of and perceived need for simulators in clinical electrophysiology: results from an EHRA physician survey.

AIMS: Simulator training has been recently introduced in electrophysiology (EP) programmes in order to improve catheter manipulation skills without complication risks. The aim of this study is to survey the current use of EP simulators and the perceived need for these tools in clinical training and practice. METHODS AND RESULTS: A 20-item online questionnaire developed by the Scientific Initiatives Committee of the European Heart Rhythm Association (EHRA) in collaboration with EHRA Digital Committee was disseminated through the EHRA Scientific Research Network members, national EP groups, and social media platforms. Seventy-four respondents from 22 countries (73% males; 50% under 40 years old) completed the survey. Despite being perceived as useful among EP professionals (81%), EP simulators are rarely a part of the institutional cardiology training programme (20%) and only 18% of the respondents have an EP simulator at their institution. When available, simulators are mainly used in EP to train transseptal puncture, ablation, and mapping, followed by device implantation (cardiac resynchronization therapy [CRT], leadless, and conduction system pacing [CSP]). Almost all respondents (96%) believe that simulator programmes should be a part of the routine institutional EP training, hopefully developed by EHRA, in order to improve the efficacy and safety of EP procedures and in particular CSP 58%, CRT 42%, leadless pacing 38%, or complex arrhythmia ablations (VT 58%, PVI 45%, and PVC 42%). CONCLUSION: This current EHRA survey identified a perceived need but a lack of institutional simulator programme access for electrophysiologists who could benefit from it in order to speed up the learning curve process and reduce complications of complex EP procedures.

Periodic verification of results' comparability between several analyzers: experience in the application of the EP31-A-IR guideline.

OBJECTIVES: To assess the usefulness of the EP31-A-IR guideline published by the Clinical and Laboratory Standards Institute (CLSI) to perform the periodic verification of results' comparability between several analyzers. METHODS: Twenty-four biochemistry parameters that could be measured in different analyzers were included: albumin, alkaline phosphatase, alanine aminotransferase, amylase, aspartate aminotransferase, calcium, chloride, C-reactive protein, creatine kinase, creatinine, direct bilirubin, gamma glutamyl transferase, glucose, lactate dehydrogenase, magnesium, phosphate, potassium, sodium, total bilirubin, total cholesterol, total protein, triglycerides, urea and uric acid. In accordance with the EP31-A-IR guideline: (1) Patient samples were selected considering the concentration or activity of interest. (2) Acceptance criteria were established specifically for each concentration or activity level. A quality specification based on biological variation or on state of the art was selected, considering the analytical performance of the available technology. (3) Maximum allowable differences (MAD) between analyzers were calculated. (4) Measurements were performed as stated in appendix B of the guideline. (5) Maximum differences between analyzers were calculated. Results were considered comparable when the maximum difference was less than or equal to the MAD. RESULTS: For the 24 parameters evaluated, any difference between analyzers exceeded the MAD. CONCLUSIONS: The EP31-A-IR guideline proved to be useful for periodic verification of results' comparability. However, it must be considered that, to be practicable, it may require to adjust the acceptance criteria in accordance to the analytical performance of the available technology; as well as the number of analytical measurements conforming to the laboratory resources.