Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50=12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity

A four-helix bundle stores copper for methane oxidation

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO). Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location

Patterns of adiposity, vascular phenotypes and cognitive function in the 1946 British Birth Cohort.

BACKGROUND: The relationship between long-term exposure to whole body or central obesity and cognitive function, as well as its potential determinants, remain controversial. In this study, we assessed (1) the potential impact of 30 years exposure to different patterns of whole body and central adiposity on cognitive function at 60-64 years, (2) whether trajectories of central adiposity can provide additional information on later cognitive function compared to trajectories of whole body adiposity, and (3) the influence of vascular phenotypes on these associations. METHODS: The study included 1249 participants from the prospective cohort MRC National Survey of Health and Development. Body mass index (BMI), waist circumference (WC), and vascular (carotid intima-media thickness, carotid-femoral pulse wave velocity) and cognitive function (memory, processing speed, reaction time) data, at 60-64 years, were used to assess the associations between different patterns of adult WC or BMI (from 36 years of age) and late midlife cognitive performance, as well as the proportion of this association explained by cardiovascular phenotypes. RESULTS: Longer exposure to elevated WC was related to lower memory performance (p < 0.001 for both) and longer choice reaction time (p = 0.003). A faster gain of WC between 36 and 43 years of age was associated with the largest change in reaction time and memory test (P < 0.05 for all). Similar associations were observed when patterns of WC were substituted with patterns of BMI, but when WC and BMI were included in the same model, only patterns of WC remained significantly associated with cognitive function. Participants who dropped one BMI category and maintained a lower BMI had similar memory performance to those of normal weight during the whole follow-up. Conversely, those who dropped and subsequently regained one BMI category had a memory function similar to those with 30 years exposure to elevated BMI. Adjustment for vascular phenotypes, levels of cardiovascular risk factors, physical activity, education, childhood cognition and socioeconomic position did not affect these associations. CONCLUSIONS: Longer exposure to elevated WC or BMI and faster WC or BMI gains between 36 and 43 years are related to lower cognitive function at 60-64 years. Patterns of WC in adulthood could provide additional information in predicting late midlife cognitive function than patterns of BMI. The acquisition of an adverse cardiovascular phenotype associated with adiposity is unlikely to account for these relationships

Evaluating spatial disparities of rotor sites and high dominant frequency regions during catheter ablation for PersAF patients targeting high dominant frequency sites using non-contacting mapping

Purpose: Several studies have emphasised the significance of high dominant frequency (HDF) and rotors in the perpetuation of AF. However, the co-localisation relationship between both attributes is not completely understood yet. In this study, we aim to evaluate the spatial distributions of HDF regions and rotor sites within the left atrium (LA) pre and post HDF-guided ablation in PersAF. Methods: This study involved 10 PersAF patients undergoing catheter ablation targeting HDF regions in the LA. 2048-channels of atrial electrograms (AEG) were collected pre- and post-ablation using a non-contact array (EnSite, Abbott). The dominant frequency (DF, 4-10 Hz) areas with DF within 0.25 Hz of the maximum out of the 2048 points were defined as "high" DF (HDF). Rotors were defined as PSs that last more than 100 ms and at a similar location through subsequent phase frames over time. Results: The results indicated an extremely poor spatial correlation between the HDF regions and sites of the rotors in pre-versus post-ablation cases for the non-terminated (pre: CORR; 0.05 ± 0.17. vs. post: CORR; -0.030 ± 0.19, and with terminated patients (pre: CORR; -0.016 ± 0.03. post: CORR; -0.022 ± 0.04). Rotors associated with AF terminations had a long-lasting life-span post-ablation (non-terminated vs. terminated 120.7 ± 6.5 ms vs. 139.9 ± 39.8 ms), high core velocity (1.35 ± 1.3 mm/ms vs. 1.32 ± 0.9 mm/ms), and were less meandering (3.4 ± 3.04 mm vs. 1.5 ± 1.2 mm). Although the results suggest a poor spatial overlapping between rotors' sites and sites of AFCL changes in terminated and non-terminated patients, a higher correlation was determined in terminated patients (spatial overlapping percentage pre: 25 ± 4.2% vs. 17 ± 3.8% vs. post: 8 ± 4.2% vs. 3.7 ± 1.7% p < 0.05, respectively). Conclusion: Using non-contact AEG, it was noted that the correlation is poor between the spatial distribution of HDF regions and sites of rotors. Rotors were longer-lasting, faster and more stationary in patients with AF termination post-ablation. Rotors sites demonstrated poor spatial overlapping with sites of AFCL changes that lead to AF termination.fals

Simultaneous Whole-Chamber Non-contact Mapping of Highest Dominant Frequency Sites During Persistent Atrial Fibrillation: A Prospective Ablation Study

PURPOSE: Sites of highest dominant frequency (HDF) are implicated by many proposed mechanisms underlying persistent atrial fibrillation (persAF). We hypothesized that prospectively identifying and ablating dynamic left atrial HDF sites would favorably impact the electrophysiological substrate of persAF. We aim to assess the feasibility of prospectively identifying HDF sites by global simultaneous left atrial mapping. METHODS: PersAF patients with no prior ablation history underwent global simultaneous left atrial non-contact mapping. 30 s of electrograms recorded during AF were exported into a bespoke MATLAB interface to identify HDF regions, which were then targeted for ablation, prior to pulmonary vein isolation. Following ablation of each region, change in AF cycle length (AFCL) was documented (≥ 10 ms considered significant). Baseline isopotential maps of ablated regions were retrospectively analyzed looking for rotors and focal activation or extinction events. RESULTS: A total of 51 HDF regions were identified and ablated in 10 patients (median DF 5.8Hz, range 4.4-7.1Hz). An increase in AFCL of was seen in 20 of the 51 regions (39%), including AF termination in 4 patients. 5 out of 10 patients (including the 4 patients where AF termination occurred with HDF-guided ablation) were free from AF recurrence at 1 year. The proportion of HDF occurrences in an ablated region was not associated with change in AFCL (τ = 0.11, p = 0.24). Regions where AFCL decreased by 10 ms or more (i.e., AF disorganization) after ablation also showed lowest baseline spectral organization (p < 0.033 for any comparison). Considering all ablated regions, the average proportion of HDF events which were also HRI events was 8.0 ± 13%. Focal activations predominated (537/1253 events) in the ablated regions on isopotential maps, were modestly associated with the proportion of HDF occurrences represented by the ablated region (Kendall's τ = 0.40, p < 0.0001), and very strongly associated with focal extinction events (τ = 0.79, p < 0.0001). Rotors were rare (4/1253 events). CONCLUSION: Targeting dynamic HDF sites is feasible and can be efficacious, but lacks specificity in identifying relevant human persAF substrate. Spectral organization may have an adjunctive role in preventing unnecessary substrate ablation. Dynamic HDF sites are not associated with observable rotational activity on isopotential mapping, but epi-endocardial breakthroughs could be contributory.fals

Are atrial fibrillation highest dominant frequency (HDF) areas the source of dominant excitation patterns? A left atrial panoramic view

Atrial fibrillation (AF) catheter ablation success depends on the possibility to accurately determine areas on the atrial endocardium at which AF activation originates. One way to determine if major AF activation pathways originate at identified source is through causality analysis. This work assessed to what extent left atrial highest dominant frequency (HDF) areas can be identified as sources of activation pathways in 10 male subjects suffering from persistent AF. Virtual electrograms were collected from 64 endocardial locations for at least 5 minutes. Frequency and causality were analyzed on 4 s signal segments Causality was assessed using the directed transfer function (DTF) algorithm, and AF activation sources were identified as endocardial locations of which the VEGM signal had high influence on other VEGM signals. Co-localization of high influence and HDF areas was evaluated for different area overlap and spectral organisation (OI) thresholds. Results show that, on average, good overlap only existed in 64.6% (± 8.8%) over all subject using the lowest threshold settings. Good overlap rates reduced with more conservative thresholds. This indicates that HDF areas might not always identify origins of main AF activation pathways

Dynamic behavior of rotors during human persistent atrial fibrillation as observed using non-contact mapping

Rotors have been related to atrial fibrillation (AF) maintenance. We analyzed the behavior of rotors in persistent AF (persAF) utilizing a novel non-contact methodology and compared this to real time dominant frequency (DF) analysis. 2048 noncontact virtual unipolar atrial electrograms (VEGMs) were collected simultaneously (EnSite Array, St. Jude Medical) from 10 persAF patients (duration: 34 ± 25 months) undergoing left atrial (LA) ablation. After QRST-removal, FFT was used to identify the global DF of the LA (range 4-10 Hz; 1 s time-window; 50 % overlap; highest DF (HDF) (DF -0.25 Hz); up to 20 s/patient). The organization index (OI) was measured and phase was found via Hilbert-transform. Phase singularities (PSs) were tracked and were categorized according to their lifespan into short (lifespan 100 ms). A total of 4578 PSs were tracked. 5.05 % (IQR: 2.75 ~ 30.25 %) of the tracked PSs were long-lived and were observed in 11 % (IQR: 2.75 ~ 17.5 %) of the windows. The windows with rotors showed significantly higher HDF (mean ± SD, 8.0 ± 0.43 Hz vs 7.71 ± 0.50 Hz, p<; 0.0001) and lower OI (0.76 ± 0.04 vs 0.79 ± 0.03, p<; 0.0001) when compared with the short-lived PSs windows. During persAF, the LA showed distinct behaviors as characterized by rotors. Often, no rotors were observed during sustained AF and, when present, the rotors continually switched between organized and disorganized behaviors. Long-lived rotors correlated with higher atrial rates. Our results suggest that rotors are not the sole perpetuating mechanism in persAF

Propagation of meandering rotors surrounded by areas of high dominant frequency in persistent atrial fibrillation

Background: Identification of arrhythmogenic regions remains a challenge in persistent atrial fibrillation (persAF). Frequency and phase analysis allows identification of potential ablation targets. Objective: This study aimed to investigate the spatiotemporal association between dominant frequency (DF) and reentrant phase activation areas. Methods: A total of 8 persAF patients undergoing first-time catheter ablation procedure were enrolled. A noncontact array catheter was deployed into the left atrium (LA) and 2048 atrial fibrillation electrograms (AEGs) were acquired for 15 seconds following ventricular far-field cancellation. DF and phase singularity (PS) points were identified from the AEGs and tracked over consecutive frames. The spatiotemporal correlation of high DF areas and PS points was investigated, and the organization index at the core of high-DF areas was compared with that of their periphery. Results: The phase maps presented multiple simultaneous PS points that drift over the LA, with preferential locations. Regions displaying higher PS concentration showed a degree of colocalization with DF sites, with PS and DF regions being neighbors in 61.8% and with PS and DF regions overlapping in 36.8% of the time windows. Sites with highest DF showed a greater degree of organization at their core compared with their periphery. After ablation, the PS incidence reduced over the entire LA (36.2% ± 23.2%, P < .05), but especially at the pulmonary veins (78.6% ± 22.2%, P < .05). Conclusion: Multiple PS points drifting over the LA were identified with their clusters correlating spatially with the DF regions. After pulmonary vein isolation, the PS’s complexity was reduced, which supports the notion that PS sites represent areas of relevance to the atrial substrate

Investigation on recurrent high dominant frequency spatiotemporal patterns during persistent atrial fibrillation

Atrial regions hosting dominant frequency (DF) may represent potential drivers of persistent atrial fibrillation (persAF). Previous work showed that DF can exhibit cyclic behaviour. This study aims to better understand the spatiotemporal behaviours of persAF over longer time periods. 10 patients undergoing persAF ablation targeted at DF were included. Left atrial (LA) non-contact virtual electrograms (VEGMs, Ensite Array, St Jude Medical) were collected for up to 5 min pre-/post- ablation. DF was identified as the peak from 4-10 Hz, in 4 s windows (50 % overlap). High DF (HDF) map was created and automated pattern recognition algorithm was applied to look for recurring HDF spatial patterns within each patient. Recurring HDF patterns were found in all patients. Patients who changed rhythm to atrial flutter after ablation demonstrated single dominant pattern (DP) among the recorded time period, which might consistent with the higher level of regularity during flutter. Ablation regularized AF as demonstrated by increased DP recurrence after ablation. The time interval (median [IQR]) of DP recurrence for the patients still in atrial fibrillation(AF) after ablation (7 patients) decreased from 21.1 s [11.8~49.7 s] to 15.7s [6.5~18.2 s]. The proposed method quantifies the spatiotemporal regularity of HDF DPs over long time periods and may offer a more comprehensive dynamic overview of persAF behaviour and the impact of ablation

Persistent atrial fibrillation hierarchical activation: from highest DF sites to wave fractionation at the boundaries

Preclinical studies showed a relationship between high dominant frequency areas (HDFA) and wave fractionation, but evidence in patient who atrial fibrillation (AF) persists for long-term periods (persAF) it is not well defined. This study aims to assess the spatiotemporal organization characteristics at HDFAs is persAF and its impact after per standard pulmonary vein isolation (PVI). Eight persAF patients had a non-contact array catheter deployed into the left atrium to collect up to 2048 AF electrograms (AEG) for 15 s. AEGs were band-pass filtered (3-30 Hz) followed by ventricular farfield cancellation. DF between 4-10 Hz and its respective organization index (OI) were calculated (4 s with 50% overlap) to produce 3D DF and OI maps. HDFA defined as the regions within a 0.25 Hz drop from the highest DF were determined and their centre of gravity (CG) calculated. Highest DF sites showed a higher OI at their core when compared to the periphery (0.422±0.101 vs. 0.386±0.126, p=0.02) and increased again organization at sites distant from the HDFAs. Similarly, after PVI, OI remained higher as compared to their periphery (0.372±0.026 vs. 0.332±0.036, p=0.22), but with significant lower values when compared with baseline (p<0.0001). PersAF patients showed higher organization in the HDFAs core when compared with its periphery