Critical Sites Research Articles

Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation.

While defective α-synuclein homeostasis is central to Parkinson's pathogenesis, fundamental questions about its degradation remain unresolved. We have developed a bimolecular fluorescence complementation assay in living cells to monitor de novo ubiquitination of α-synuclein and identified lysine residues 45, 58, and 60 as critical ubiquitination sites for its degradation. This is mediated by NBR1 binding and entry into endosomes in a process that involves ESCRT I-III for subsequent lysosomal degradation. Autophagy or the autophagic chaperone Hsc70 is dispensable for this pathway. Antibodies against diglycine-modified α-synuclein peptides confirmed that endogenous α-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated α-synuclein was detected in Lewy bodies and cellular models of aggregation, suggesting that it may be entrapped with endo/lysosomes in inclusions. Our data elucidate the intracellular trafficking of de novo ubiquitinated α-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.

Long-Term Changes in a Pygmy Cormorant Microcarbo pygmaeus Wintering Population in Southern Bulgaria

Waterbirds are declining globally due to different threats that affect their abundance and shift both their breeding and wintering ranges. Cormorants disperse over vast distances during winter and are suitable indicators of the impacts of human-wildlife interactions and abiotic factors. During wintering periods, Pygmy Cormorants Microcarbo pygmaeus use regular roosts where they overnight on suitable perches, a refuge from adverse weather conditions and disturbances. Our study aimed to determine the wintering population size of the Pygmy Cormorant and its dynamics across 23-years (1996–2019) at seven roosting sites in the Maritsa River valley, Southern Bulgaria. This is a critical site for the species conservation where 3065–8180 birds are wintering annually. The wintering Pygmy Cormorant population showed a tendency to increase between 1996 and 2019 (λ = 1.03 ± 0.001). Each year Pygmy Cormorants were most abundant in December (mean ± SE, 2609 ± 185 individuals), while the lowest numbers were recorded in March (1207 ± 106). Pygmy Cormorants aggregated to roost between 4:00 PM and 5:30 PM throughout the study. The area of wintering foraging habitat of the Pygmy Cormorant increased significantly across all roosting sites in the study period from 2675.97 ha to 22564.35 ha. The number of Pygmy Cormorants at the roost was positively affected by the drop in the daily air mean temperatures and the decrease of the day length. Despite the global population increase, the Pygmy Cormorant faces a high risk of human-induced mortality during wintering and is thus regionally classified as endangered.

The hypothalamus as a key regulator of glucose homeostasis: emerging roles of the brain renin-angiotensin system.

The regulation of plasma glucose levels is a complex and multifactorial process involving a network of receptors and signaling pathways across numerous organs that act in concert to ensure homeostasis. However, much about the mechanisms and pathways by which the brain regulates glycemic homeostasis remains poorly understood. Understanding the precise mechanisms and circuits employed by the central nervous system to control glucose is critical to resolving the diabetes epidemic. The hypothalamus, a key integrative center within the central nervous system, has recently emerged as a critical site in the regulation of glucose homeostasis. Here, we review the current understanding of the role of the hypothalamus in regulating glucose homeostasis, with an emphasis on the paraventricular nucleus, the arcuate nucleus, the ventromedial hypothalamus, and lateral hypothalamus. In particular, we highlight the emerging role of the brain renin-angiotensin system in the hypothalamus in regulating energy expenditure and metabolic rate, as well as its potential importance in the regulation of glucose homeostasis.

EvdS6 is a bifunctional decarboxylase from the everninomicin gene cluster

The everninomicins are bacterially produced antibiotic octasaccharides characterized by the presence of two interglycosidic spirocyclic ortho-δ-lactone (orthoester) moieties. The terminating G- and H-ring sugars, L-lyxose and C-4 branched sugar β-D-eurekanate, are proposed to be biosynthetically derived from nucleotide diphosphate pentose sugar pyranosides; however, the identity of these precursors and their biosynthetic origin remain to be determined. Herein we identify a new glucuronic acid decarboxylase from Micromonospora belonging to the superfamily of short-chain dehydrogenase/reductase enzymes, EvdS6. Biochemical characterization demonstrated that EvdS6 is an NAD+-dependent bifunctional enzyme that produces a mixture of two products, differing in the sugar C-4 oxidation state. This product distribution is atypical for glucuronic acid decarboxylating enzymes, most of which favor production of the reduced sugar and a minority of which favor release of the oxidized product. Spectroscopic and stereochemical analysis of reaction products revealed that the first product released is the oxidatively produced 4-keto-D-xylose and the second product is the reduced D-xylose. X-ray crystallographic analysis of EvdS6 at 1.51Å resolution with bound co-factor and TDP demonstrated that the overall geometry of the EvdS6 active site is conserved with other SDR enzymes and enabled studies probing structural determinants for the reductive half of the net neutral catalytic cycle. Critical active site threonine and aspartate residues were unambiguously identified as essential in the reductive step of the reaction and resulted in enzyme variants producing almost exclusively the keto sugar. This work defines potential precursors for the G-ring L-lyxose and resolves likely origins of the H-ring β-D-eurekanate sugar precursor.

Targeting Trop2 by Bruceine D suppresses breast cancer metastasis by blocking Trop2/β-catenin positive feedback loop

IntroductionTumor-associated calcium signal transducer 2 (Trop2) has been used as a transport gate for cytotoxic agents into cells in antibody–drug conjugate designs because of its expression in a wide range of solid tumors. However, the specific role of Trop2 itself in breast cancer progression remains unclear and small molecules targeting Trop2 have not yet been reported. ObjectivesTo screen small molecules targeting Trop2, and to reveal its pharmacological effects and the molecular mechanisms of action. MethodsSmall molecule targeting Trop2 was identified by cell membrane chromatography, and validated by cellular thermal shift assay and point mutation analyses. We investigated the pharmacological effects of Trop2 inhibitor using RNA-seq, human foreskin fibroblast (HFF)-derived extracellular matrix (ECM), Matrigel drop invasion assays, colony-forming assay, xenograft tumor model, 4T1 orthotopic metastasis model and 4T1 experimental metastasis model. The molecular mechanism was determined using immunoprecipitation, mass spectrometry, immunofluorescence, immunohistochemistry and Western blotting. ResultsHere we identified Bruceine D (BD) as the inhibitor of Trop2, and demonstrated anti-metastasis effects of BD in breast cancer. Notably, Lys307 and Glu310 residues of Trop2 acted as critical sites for BD binding. Mechanistically, BD suppressed Trop2-induced cancer metastasis by blocking the formation of Trop2/β-catenin positive loop, in which the Trop2/β-catenin complex prevented β-catenin from being degraded via the ubiquitin-proteosome pathway. Destabilized β-catenin caused by BD reduced nucleus translocation, leading to the reduction of transcription of Trop2, the reversal of epithelial-mesenchymal transition (EMT) process, and the inhibition of ECM remodeling, further inhibiting cancer metastasis. Additionally, the inhibitory effects of BD on lung metastatic colonization and the beneficial effects of BD on prolongation of survival were validated in 4T1 experimental metastasis model. ConclusionsThese results support the tumor-promoting role of Trop2 in breast cancer by stabilizing β-catenin in Trop2/β-catenin positive loop, and suggest Bruceine D as a promising candidate for Trop2 inhibition.

Evaluation of bleeding risk between apixaban and rivaroxaban in patients with gastrointestinal cancer.

e16323 Background: Patients with cancer are at a higher risk for developing venous thromboembolisms (VTEs), leading to further complications. The National Comprehensive Cancer Network (NCCN) guidelines currently recommend using low-molecular-weight heparin (LMWH) or direct oral anticoagulants (DOACs) for the treatment of VTEs in oncology patients. Other trials compare DOACs with LMWH to determine the risk of bleeding. The Select-D trial found that rivaroxaban had a higher risk of bleeding in patients with gastrointestinal (GI) cancer as compared to LMWH. However, the Caravaggio trial found that apixaban had no increase of bleeding for patients with GI cancer. Due to the inherent risk of major bleeding shown in the subgroup analysis of the trials above, the guidelines recommend LMWH as the preferred agent for patients with GI cancers. At Winship Cancer Institute, DOACs are used for the treatment of cancer-associated VTE for patients with GI malignancies. The purpose of this study was to assess real-world safety outcomes of patients with GI cancer who were treated with apixaban or rivaroxaban for VTE. Methods: This study is a single-center, retrospective chart review of patients 18 years or older with GI cancer receiving treatment for VTE with either apixaban or rivaroxaban. The study looks to review bleeding outcomes in patients who started their VTE treatment from January 1, 2017 – December 31, 2021. The primary outcome of this study was to evaluate the rates of major bleeding between apixaban and rivaroxaban. Major bleeding was defined as a decrease in hemoglobin of ≥ 2g/dL over 24 hours, transfusion of ≥2 units of red blood cells, bleeding that requires surgical intervention, or bleeding at critical sites. Secondary outcomes included clinically relevant non-major bleeding (CRNMB) defined by bleeding compromising hemodynamics, hematemesis, hemoptysis, rectal bleeding, or hematuria, VTE recurrence, and death from bleeding. Data collected to measure these outcomes included patient characteristics such as renal function, weight, primary cancer site, medication dosing, and duration and indication of treatment. Results: A total of 300 patients qualified for inclusion of this study. Of these, 89 were in the apixaban group and 211 in the rivaroxaban group. Major bleeding occurred in 3 patients in the apixaban group (3.4%) and 18 in the rivaroxaban group (8.5%). CRNMB occurred in 2 patients in the apixaban group (2.2%) and 16 patients in the rivaroxaban group (7.6%). VTE recurrence occurred in 4 patients in the apixaban group (4.5%) and 9 patients in the rivaroxaban group (4.3%). Conclusions: This is the only study to date to evaluate DOACs head to head and their bleeding risk in cancer patients. Apixaban was associated with lower rates of major bleeds and CRNMB, but relatively similar rates of VTE recurrence. The results of this study warrant consideration of using DOACs for patients with GI cancers in the upfront setting.

Submonolayer Cu/Pt film as bifunctional catalyst for CO oxidation: An STM study

Considering the high activity in CO oxidation as well as the related reactions, the surface structure of Cu/Pt bimetallic system and its interaction with CO and O2 molecules have been revisited with low-temperature scanning tunneling microscopy (STM) in combination with density-functional theory calculations (DFT). The extensively concerned Cu/Pt subsurface alloy (Cu-SSA) is clearly demonstrated with relative deactivation to CO and drastically weakened binding to O. In contrast, the submonolayer Cu film on Pt(111) manifests a significant enhancement in activating the O2 molecules while reserving the strong binding to CO at the uncovered Pt area. Detailed control experiments corroborated by DFT calculations revealed that the Cu/Pt interface serves as the critical sites for both O2 activation and the CO oxidation reaction. Therefore, the submonolayer Cu film may register a more promising catalyst structure than the Cu-SSA configuration for the Cu/Pt bimetallic system.

Neutralisation of SARS-CoV-2 by monoclonal antibody through dual targeting powder formulation.

Neutralising monoclonal antibody (mAb) is an important weapon in our arsenal for combating respiratory viral infections. However, the effectiveness of neutralising mAb has been impeded by the rapid emergence of mutant variants. Early administration of broad-spectrum mAb with improved delivery efficiency can potentially enhance efficacy and patient outcomes. WKS13 is a humanised mAb which was previously demonstrated to exhibit broad-spectrum activity against SARS-CoV-2 variants. In this study, a dual targeting formulation strategy was designed to deliver WKS13 to both the nasal cavity and lower airways, the two critical sites of infection caused by SARS-CoV-2. Dry powders of WKS13 were first prepared by spray drying, with cyclodextrin used as stabiliser excipient. Two-fluid nozzle (TFN) was used to produce particles below 5μm for lung deposition (C-TFN formulation) and ultrasonic nozzle (USN) was used to produce particles above 10μm for nasal deposition (C-USN formulation). Gel electrophoresis and size exclusion chromatography studies showed that the structural integrity of mAb was successfully preserved with no sign of aggregation after spray drying. To achieve dual targeting property, C-TFN and C-USN were mixed at various ratios. The aerosolisation property of the mixed formulations dispersed from a nasal powder device was examined using a Next Generation Impactor (NGI) coupled with a glass expansion chamber. When the ratio of C-TFN in the mixed formulation increased, the fraction of particles deposited in the lung increased proportionally while the fraction of particles deposited in the nasal cavity decreased correspondingly. A customisable aerosol deposition profile could therefore be achieved by manipulating the mixing ratio between C-TFN and C-USN. Dual administration of C-TFN and C-USN powders to the lung and nasal cavity of hamsters, respectively, was effective in offering prophylactic protection against SARS-CoV-2 Delta variant. Viral loads in both the lung tissues and nasal wash were significantly reduced, and the efficacy was comparable to systemic administration of unformulated WKS13. Overall, dual targeting powder formulation of neutralising mAb is a promising approach for prophylaxis of respiratory viral infections. The ease and non-invasive administration of dual targeting nasal powder may facilitate the widespread distribution of neutralising mAb during the early stage of unpredictable outbreaks.

Differential recognition of influenza A virus H1N1 neuraminidase by DNA vaccine-induced antibodies in pigs and ferrets.

Neuraminidase (NA) accounts for approximately 10-20% of the total glycoproteins on the surface of influenza viruses. It cleaves sialic acids on glycoproteins, which facilitates virus entry into the airways by cleaving heavily glycosylated mucins in mucus and the release of progeny virus from the surface of infected cells. These functions make NA an attractive vaccine target. To inform rational vaccine design, we define the functionality of influenza DNA vaccine-induced NA-specific antibodies relative to antigenic sites in pigs and ferrets challenged with a vaccine-homologous A/California/7/2009(H1N1)pdm09 strain. Sera collected pre-vaccination, post-vaccination and post-challenge were analyzed for antibody-mediated inhibition of NA activity using a recombinant H7N1CA09 virus. Antigenic sites were further identified with linear and conformational peptide microarrays spanning the full NA of A/California/04/2009(H1N1)pdm09. Vaccine-induced NA-specific antibodies inhibited the enzymatic function of NA in both animal models. The antibodies target critical sites of NA such as the enzymatic site, second sialic binding site and framework residues, shown here by high-resolution epitope mapping. New possible antigenic sites were identified that potentially block the catalytic activity of NA, including an epitope recognized solely in pigs and ferrets with neuraminidase inhibition, which could be a key antigenic site affecting NA function. These findings show that our influenza DNA vaccine candidate induces NA-specific antibodies that target known critical sites, and new potential antigenic sites of NA, inhibiting the catalytic activity of NA.

0073 Investigating the epigenetic mechanisms underlying memory consolidation during sleep

Abstract Introduction It has been established that sleep after learning something (“post-learning sleep”) actively consolidates what was learned. Yet, the underlying cellular and molecular mechanisms are undergoing active investigation. The fear conditioning (FC) memory paradigm, in which an animal learns to associate an environmental cue to an aversive foot shock, has been widely used for memory investigation. While amygdala and hippocampus are two critical sites for processing and storing the FC memory, how the amygdala consolidate this memory during sleep, from both cellular and molecular perspectives, has been little understood, compared to the hippocampus. Moreover, one potential molecular substrate of memory, DNA methylation, has been studied in the hippocampus for its role in learning and memory. Yet it is little known for its memory role in the BLA. Methods Here, we used fiber photometry, miniscope calcium imaging, EEG recording, optogenetic manipulation, and whole genome bisulfite sequencing to study the neural mechanisms underlying memory consolidation in the basolateral amygdala (BLA) during sleep. Results (1) We found that the BLA neurons display increased activity during REM sleep, compared to that in non-REM (NREM) sleep. (2) Using closed-loop optogenetic strategy, our preliminary results showed that silencing BLA activity during REM sleep leads to memory impairment in contextual fear conditioning task. (3) We compared DNA methylation of BLA neurons at a whole genome level between mice with and without sleep after FC learning and identified differentially methylated regions between them. Conclusion Together, our data suggests that the BLA activity during REM sleep is required for consolidation of fear memory, and DNA methylation might mediate this process. Support (if any)

Long term results of catheter ablation in patients presented with ventricular storm

Abstract Funding Acknowledgements Type of funding sources: None. Background and Objective Catheter ablation is a treatment option in patients with ventricular tachycardia (VT storm, especially when refractory to medical treatment. In this trial, we aimed to evaluate the long-term results of catheter ablation in patients with VT storm. Method Between January 2015 and January 2022T, we enrolled a total of 65 VT storm patients who underwent catheter ablation. All patients who have either documented episodes of VT or appropriate ICD shocks more than 3in the last 24 hours. Medical treatment with antiarrhythmic drugs was given to all patients before catheter ablation. After the procedure, all-cause mortality, hospitalization due to stroke/myocardial infarction/heart failure, ICD shocks, and anti-tachycardia pacing therapies were recorded during follow-up. Results Among the whole study population (the median age is 65 years (17-85); 56 (86.2%) males) 37 (56.9%) had ischemic CMP, 24 (36.9%) had non-ischemic CMP whose 8 were ARVD, and 3 (4.6%) HCMP. The median left ventricle ejection fraction was 30% (12 – 65%). Activation mapping was performed in 60 patients, and the critical isthmus was ablated in 60 patients whose 22 had more than one critical ablation site. In 3 patients, only substrate mapping was performed, and substrate homogenization was done. In 2 patients, premature ventricular ectopy, which inducted VT, ablation was performed. Epicardial mapping was performed in 23 patients (35.4%). The median follow-up duration was 23 months (1-94 months). During follow-up, 23 patients died, 43 were hospitalized, and four presented again with VS storm. In 13 patients, appropriate shock/pacing therapy was observed. After the index procedure, re-do catheter ablation of ventricular tachycardia was performed in 8 patients. The mean postprocedural VT-free survival time was estimated at 76.5 months (SE:5.0, 95% CI: 66.6-86.4). Conclusion Catheter ablation therapy is an effective option for both acute and long-term management of patients with VT storms. However, postprocedural mortality and morbidity remain high in long-term follow-up duration. Long-term results may be related to the comorbidities and nature of underlying cardiac disorders.

Efficacy of functional substrate map-guide approach for wide circumferential pulmonary vein isolation

Abstract Funding Acknowledgements Type of funding sources: None. Background Pulmonary vein isolation (PVI) with anatomical approach targeting closer interlesion distance (ILD) would be effective, but not efficient. A functional substrate map (FSM) can reveal the unnecessary application site including physiological block and the critical ablation site including direct epicardial connections. Purpose We investigated the efficacy of FSM-guide approach for wide circumferential PVI. Methods We enrolled 55 consecutive patients underwent radiofrequency PVI for atrial fibrillation. In earlier 27 patients were ablated by anatomical approach with closer ILD (<5mm) (control group), in latter 28 patients were by real time PV electrogram and high density electro-anatomical map guide (FSM-guide group). In FSM-guide group, characteristics of electrical conduction including voltage, direction , velocity and barrier between PV and left atrium and epicardial connection were assessed by local activation, coherent and ripple map before ablation. Distributions of the segments required or unnecessary ablation for PVI completion were examined. Results The success rate of first-pass isolation was higher in FSM-guide group than in control (96.4% vs. 74.1%). The number of required ablation tags for PVI were significantly lesser in FSM-guide group than control group (left-sided PV (LPV); 23.2±1.6 vs. 29.2±1.6, p=0.0112, right-sided PV (RPV); 22.6±1.7 vs. 32.4±1.8, p=0.0124, respectively). The time for PVI completion was significantly shorter in FSM-guide group than control group (RPV; 17.0±2.2 min vs. 25.2±2.8 min, p=0.0124, LPV; 18.7±1.5 min vs. 20.5±1.5 min, p=0.042, respectively). In FSM-guide group, unnecessary application sites were frequently located at posterior of RIPV (46%), anterior of LIPV (43%) and anterior of RSPV (35.7%). Frequently required segments for PVI completion were roof and carina of LPV and roof and posterior of RPV. In most cases of control group, full circumferential ablation was required (LPV 22/27(81%) and RPV 24/27(89%)). Acute reconnections during procedure were similar in FSM-guide group and control group (LPV; 6/28 (21.4%) vs. 4/27 (14.8%), p=0.7287, RPV; 9/28 (32.1%) vs. 4/27 (14.8%), p=0.2047, respectively). All of reconnection sites in FSM-guide group were ablated site. Conclusions FSM-guide approach for extensive PVI could reveal unnecessary application sites and result in shorter procedure with comparable ablation quality.

Accuracy of a novel ECGi system in localising the critical isthmus exit site in scar-dependant re-entrant ventricular tachycardia

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Vision in ventricular onset (VIVO) is a novel, non-invasive, mapping system designed to locate the site of earliest activation in ventricular arrhythmia. VIVO utilises a mathematical algorithm, cross-sectional imaging data (CT or MRI), 3D images of the electrodes on the patient torso, and a 12-lead electrocardiogram (ECG) to identify the site of earliest ventricular activation.(1) The system has been shown to accurately predict earliest activation of focal premature ventricular contractions (PVCs), but its accuracy in scar dependant re-entrant ventricular tachycardia (VT) has not been investigated.(1-3) Objective To assess the accuracy of VIVO in localising the VT exit site for patients with ischaemic cardiomyopathy (ICM) and scar related re-entrant VT. Methods 5 patients with ICM (age 56 ± 19 yrs, male n = 5) were investigated (table 1). All patients had an implantable cardioverter-defibrillator with standard indications for VT ablation. Invasive electro-anatomical mapping was performed with the Advisor HD Grid multipolar catheter and maps were generated using omnipolar electrograms (EGMs). The VT exit site was identified using activation ± pace-maps and the location defined by the primary operator using the American Heart Association (AHA) 17 segment model of the left ventricle.(4) Using the intra-procedural VT ECG recordings, VIVO maps were constructed post-procedure. The VIVO predicted VT exit site was identified using the AHA 17-segment model by a second operator, blinded to the results of the procedure. A "complete match" was defined as exact segment concordance between the two operators, "partial match" as adjacent segments, and "no match" if neither requirements satisfied. Results Mean left ventricular ejection fraction was 30.5 ± 9.0%. Mean procedure time was 233 ± 31 minutes, with a mean ablation time of 24 ± 18 minutes. A total of 11 re-entrant VTs were mapped. The VT exit site was identified in all cases (10 activation-map; 1 pace-map). A complete match was seen in 91% of VTs and partial match in 9% (table 2). None of the cases demonstrated a "no match" between the invasive and VIVO maps. There were no procedural complications. Conclusion The VIVO mapping system can accurately predict the VT exit site in scar dependant re-entrant VT. Further studies to assess the feasibility of VIVO-guided ablation in scar dependant VT are ongoing.

Optimisation of decrementing evoked potential mapping for functional substrate identification in ischaemic ventricular tachycardia ablation

Abstract Funding Acknowledgements Type of funding sources: None. Background Decrement evoked potential (DeEP) mapping has been shown to identify the functional substrate critical to the Ventricular Tachycardia (VT) circuit with a higher specificity than late potential mapping.[1] However, there is limited data on methodological optimisation of this technique which could identify more relevant regions of conduction delay and functional block that occurs during clinical VT by activating the substrate at coupling intervals closer to the VT cycle length (CL). Purpose The goal of this study was to investigate a range of short coupled extra-stimuli and decrement thresholds to improve DeEP mapping target identification in patients with ischaemic cardiomyopathy. Methods Thirteen patients (70±12 years) underwent VT ablation. Mapping was performed with the Advisor HD Grid multipolar catheter. Maps were generated using omnipolar electrograms (EGMs). DeEP mapping was performed using 3 different extra-stimuli (S2) sequentially in each case: (1) 400 ms (DeEP-400); (2) VT cycle length (VTCL); (3) ventricular effective refractory period (VERP) + 20 ms. For each of the DeEP-400, DeEP-VTCL and DeEP-VERP maps, the definition of a DeEP was altered from the standard 10 ms decrement to multiple thresholds of 10 ms to 50 ms in 10 ms increments (figure 1). DeEP maps were compared to the VT exit site identified with VT activation maps. All patients underwent DeEP-400 focused ablation. Results All patients had 1 clinical VT (VTCL = 353 ± 48 ms; VERP + 20 ms = 300 ± 20 ms). Mean left ventricular ejection fraction was 30 ± 12 %. Sensitivity and specificity analyses (figure 2) show the optimal threshold for defining a DeEP is with an EGM prolongation of ≥ 20 ms for the DeEP-400 (sens. 77%, spec. 79%, ROC AUC 0.84), DeEP-VTCL (sens. 77%, spec. 79%, ROC AUC 0.83) and DeEP-VERP (sens. 69%, spec. 74%, ROC AUC 0.76) maps. Proportional to total mapped area, the DeEP-400 map area was significantly smaller than DeEP-VERP map area (19.0 ± 7.6 % vs 26.4 ± 14.3%; p=0.02). Delivering the S2 at the conventional VERP + 20 ms using a 10 ms decrement definition yielded a sensitivity of 84.6% and a specificity of 62.0% with a target ablation area of 33.9 cm2. With the optimised protocol of delivering the S2 at 400 ms using a 20 ms decrement definition, the sensitivity remains high at 76.9% with an improved specificity of 79.0%, thus reducing the ablation target area by 50% to 16.8 cm2. 92% patients were free of any VT after DeEP-400 guided ablation at 6-month median follow-up. Conclusion In this prospective, mechanistic study utilising omnipolar technology to explore a range of S2 extra-stimuli and decrement thresholds, DeEPs were optimally defined by a conduction delay of ≥ 20 ms with an S2 extra-stimulus coupling interval of 400 ms or VTCL to more accurately localise the VT exit site. This optimised DeEP mapping protocol further refines identification of the critical myocardial sites involved in the initiation and maintenance of VT.

A novel method to define critical isthmus of left atrial tachycardia; late potential mapping during sinus rhythm

Abstract Funding Acknowledgements Type of funding sources: None. Background Atrial tachycardia (AT) is a commonly encountered rhythm disorder in patients with underlying atrial scar. The role of late potential (LP) based mapping in the detection of AT critical isthmus has yet to be systematically evaluated. Purpose We aimed to investigate the relationship between the atrial LPs (ALP) localization in sinus rhythm and the critical isthmus of re-entrant ATs in patients with underlying atrial low-voltage areas. Methods Thirty-five symptomatic patients [mean age: 62 ±9, gender: 25 (71.4%) female] with left AT, who had undergone catheter ablation, were enrolled. Voltage and LP mapping were performed in sinus rhythm in both atriums. Low voltage areas and signals with continuous-fragmented morphology were tagged. Isochronal late activation mapping (ILAM) was also performed in all procedures. After induction of AT, activation mapping was performed during tachycardia in all patients to detect critical sites of the tachycardia. Results Activation mapping indicated single re-entry in 30 patients and ≥1 re-entry in 5 patients. Localized re-entry was observed in 20 (51.2%) patients, macro re-entry in 18 (46.1%), and biatrial re-entry in 1(2.5%). The mean fractionated electrogram duration in the AT termination site was calculated at 134.3±46.7 ms and the mean bipolar voltage at 0.17±0.12 mV. The ALPs were tagged in 25 (64.1%) patients on the anterior wall, 12 (34.3%) on the roof, 5 (12.8%) on the septal wall and 2 (5.7%) in the Bachmann’s Bundle region. All ALPs were in the latest activated zones of ILAM in patients with localized re-entry and predicted successful ablation sites to terminate the AT. Freedom from atrial tachyarrhythmias (ATAs) was 78.7% at six months during a mean follow-up duration of 338 ±226 days. Conclusion Our findings demonstrated the utility of ALPs during sinus rhythm to predict the successful termination sites for left ATs. These areas displayed continuous-fragmented signal morphology during sinus/paced rhythm in correlation with the critical isthmus of AT detected during activation mapping.

Conduction Velocity Dispersion Predicts Postinfarct Ventricular Tachycardia Circuit Sites and Associates With Lipomatous Metaplasia

BackgroundRegional myocardial conduction velocity (CV) dispersion has not been studied in postinfarct patients with ventricular tachycardia (VT). ObjectivesThis study sought to compare the following: 1) the association of CV dispersion vs repolarization dispersion with VT circuit sites; and 2) myocardial lipomatous metaplasia (LM) vs fibrosis as the anatomic substrate for CV dispersion. MethodsAmong 33 postinfarct patients with VT, we characterized dense and border zone infarct tissue by late gadolinium enhancement cardiac magnetic resonance, and LM by computed tomography, with both images registered with electroanatomic maps. Activation recovery interval (ARI) was the time interval from the minimum derivative within the QRS complex to the maximum derivative within the T-wave on unipolar electrograms. CV at each EAM point was the mean CV between that point and 5 adjacent points along the activation wave front. CV and ARI dispersion were the coefficient of variation (CoV) of CV and ARI per American Heart Association (AHA) segment, respectively. ResultsRegional CV dispersion exhibited a much larger range than ARI dispersion, with median 0.65 vs 0.24; P < 0.001. CV dispersion was a more robust predictor of the number of critical VT sites per AHA segment than ARI dispersion. Regional LM area was more strongly associated with CV dispersion than fibrosis area. LM area was larger (median 0.44 vs 0.20 cm2; P < 0.001) in AHA segments with mean CV <36 cm/s and CoV_CV >0.65 than those with mean CV <36 cm/s and CoV_CV <0.65. ConclusionsRegional CV dispersion more strongly predicts VT circuit sites than repolarization dispersion, and LM is a critical substrate for CV dispersion.

Queer creative Indian city: queer film festivals, precarious cultural work and community making in Kolkata

ABSTRACT In this article, we offer a new concept of the “queer creative city”, through a critical examination of how a regional queer Bengali film culture has emerged in Kolkata as a result of the convergence of certain urban policies, queer political organising and cultural activism. We explore two queer film festivals in Kolkata – the Siddharth Gautam Film Festival and Dialogues, both having a very significant impact in transforming LGBTQ + lives in Kolkata. Through archival research, autoethnography and conducting extensive interviews with organising committees, venue sponsors and owners, and viewers, we show how these film festivals and a pre-existing Bengali film culture engendered the emergence of a prolific creative queer city, which became a site of resistance, and community building that created a solid base for queer counterpublics. Queer film festivals, we argue, are critical sites for charting the dynamics of the public sphere in contemporary India.

Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.

Universities, imperialism and the collective work ahead.

Universities, imperialism and the collective work ahead.

Ferroptosis: whERe is the critical site of lipid peroxidation?

OPINION article Front. Cell Dev. Biol., 18 May 2023Sec. Cell Death and Survival Volume 11 - 2023 | https://doi.org/10.3389/fcell.2023.1179245