Specific Ablation Research Articles

Cardioprotective effects of nicorandil on mitochondrial apoptosis signaling in the failing heart of cardiac-specific Bcl-2-associated athanogene (BAG) 3 knockout mice

Abstract Background Bcl-2-associated athanogene (BAG) 3 is a known regulator of mitochondrial apoptotic cell death and autophagic protein degradation in the heart. Disruption of the BAG3 gene in a cardiac-specific manner can lead to cardiac failure in mice. However, therapeutic approaches for cardiac failure induced by BAG3 gene disruption remain poorly understood. Method and results We characterized mice with the ablated BAG3 gene using the Cre-recombinase-loxp site system in a cardiac-specific manner (BAG3f/f crossbred with cardiac specific Cre transgenic mice; BAG3 cKO) and examined the therapeutic effect of nicorandil, an antianginal drug, in BAG3 cKO mice. Cardiac BAG3 levels were markedly reduced in the cytoplasmic and mitochondrial fractions from the hearts of BAG3 cKO mice compared to BAG3f/f mice. BAG3cKO mice exhibited cardiac disease such as a reduction in fractional shortening detected by echocardiography, cardiac fibrosis at 6 months of age, and approximately 40% premature death by 6 months of age. Electron microscopy analysis confirmed cardiomyocyte loss and mitochondrial abnormalities in some areas of the heart section from BAG3cKO mice at 4.5 months of age. A marked decrease in the level of cytochrome C in the mitochondrial fraction and a slight increase in the cytoplasmic fraction, along with apoptotic cell death, were observed in hearts from BAG3 cKO mice at 4.5 months. Mitochondrial levels of anti-apoptotic factors, such as Bcl-2 and Bcl-xl, were reduced, while there was a marked increase in Bak1, an apoptotic inducible factor, in hearts from BAG3 cKO mice at 4.5 months of age. These results suggest that cardiac mitochondrial impairment and subsequent apoptotic cell death occurred in BAG3cKO mice. Nicorandil treatment (81 mg/L drinking water) initiated at 2 months of age prevented the reduction of fractional shortening, cardiac fibrosis, ultrastructural cardiac abnormalities, and premature death. Furthermore, nicorandil treatment prevented the decrease in mitochondrial cytochrome C and the increase in cytoplasmic cytochrome C in hearts from BAG3 cKO mice at 4.5 months of age. Nicorandil treatment also inhibited the elevation of mitochondrial Bak1 levels, reduction in Bcl-2 levels, and occurrence of apoptotic cell death in hearts from BAG3 cKO mice at 4.5 months of age. Conclusion Long-term treatment with nicorandil can inhibit cardiac disease induced by cardiac- specific BAG3 ablation through mitochondrial protection via the prevention of activation of mitochondrial apoptosis signaling.

High density mapping accuracy and efficiency in atrial fibrillation: real-world outcomes from the SECURE registry

Abstract Background/Introduction A high-density (HD) mapping catheter is valuable for validating pulmonary vein isolation (PVI) and analysing arrhythmic substrates of the left atrium (LA) during atrial fibrillation (AF) ablation. Multiple HD mapping catheters are available for use in clinical practice. A novel octa-spline mapping catheter with a close internal unipolar reference electrode is now available and may improve mapping efficiencies and image resolutions (Figure). Purpose This study aims to report real-world experiences with different commercially available HD mapping catheters in AF ablation and compare mapping performance. Methods SECURE is an ongoing observational, prospective, post-marketing study collecting real-world data on ablation procedures performed per standard-of-care. At the time of data extraction (Nov. 2023), 16 sites across 8 countries had enrolled 2,366 patients (pts), with 12-month follow-up data available for 828 pts. The current analysis, which included consecutive pts undergoing AF ablation, evaluated mapping efficiency and procedural outcomes with 3 HD mapping catheters: circular catheter (CC), penta-spline catheter (PSC), and octa-spline catheter (OSC). Mapping data were analysed with a cloud-based data management and artificial intelligence-powered algorithm. Results Overall, 859 pts with AF (paroxysmal AF [PAF], n=465; persistent AF [PsAF], n=240; long-standing PsAF [LS PsAF], n=154) were included in this analysis. The CC, PSC, and OSC were used in 428, 210, and 221 pts, respectively. Procedures using the OSC had a significantly higher mean mapping area (31,989.9 mm2) than those performed with the CC (23,152.2 mm2; P<0.0001) and PSC (29,804.6 mm2; P=0.0027), as well as a >3-fold higher mean mapping rate (138.4 vs 20.8 and 43.4 points/min, respectively; both P<0.0001). The higher number of mapping points translated to a higher mean mapping density (OSC, 0.34 vs CC, 0.05 and PSC, 0.07 points/mm2; both P<0.0001), with more uniform point distribution and less interpolation. With the CC, PSC, and OSC, respectively, total mean procedure times (incl PVI + other targets) were 118.0, 118.7, and 165.1 min, (P<0.0001 for OSC vs. both CC and PSC) and mean pre-ablation mapping times were 25.2, 25.0, and 21.5 min (P<0.0004 for OSC vs. both CC and PSC). The improved mapping quality and efficiency with the OSC allowed to include specific ablation targets beyond PVI. Fewer PVI-only procedures were performed with the OSC (29.4%) versus the CC (62.1%) and PSC (54.8%), while ablation of the PVI+ other targets was more common with the OSC versus the CC and PSC, including the cavotricuspid isthmus (37.1% vs 17.3% and 16.7%), LA roof (42.5% vs 13.1% and 21.4%), and LA posterior wall (21.7% vs 10.5% and 16.2%). Results were overall consistent per AF type (Table). Conclusions By providing more accurate mapping, the OSC allows for improved analysis of the arrhythmic substrate, which may improve ablation outcomes for pts with PAF and PsAF.Figure 1

Ablation therapy following unsuccessful electrical cardioversion in patients with persistent atrial fibrillation

Electrical cardioversion (ECV) a widely utilized intervention for persistent atrial fibrillation (AF) aimed at restoring sinus rhythm. However, ECV can be ineffective, raising questions about subsequent treatment options. This study aimed to compare the outcomes of non-ablation therapy versus ablation therapy following unsuccessful ECV. A total of 125 consecutive patients with persistent AF who underwent unsuccessful ECV between November 2017 and August 2023 was included in this retrospective analysis. Of these, 51.2% received only medical therapy (non-ablation therapy group, n = 64), while 48.8% underwent AF ablation (ablation therapy group, n = 61). Various ablation methods were employed, including catheter and thoracoscopic ablation. Ablation therapy was associated with significantly better AF-free survival compared to non-ablation therapy [hazard ratio (HR), 0.37; 95% confidence interval (CI) 0.22–0.61; p < 0.01]. There was no difference of AF-free survival between catheter ablation and thoracoscopic ablation groups (HR 0.79, 95% CI 0.34–1.83; p = 0.58). AF duration > 5 year (HR 1.51; 95% CI 0.930–2.437; p = 0.10), BMI ≤ 25 kg/m2 (HR 1.61; 95% CI 1.004–2.581; p = 0.05) and diabetes (HR 2.38; 95% CI 0.902–6.266; p = 0.08) were considerable as predictor of AF recurrence. Ablation therapy following unsuccessful ECV was associated with maintaining sinus rhythm, regardless of the specific ablation method utilized.

Relocalizing transcriptional kinases to activate apoptosis.

Kinases are critical regulators of cellular function that are commonly implicated in the mechanisms underlying disease. Most drugs that target kinases are molecules that inhibit their catalytic activity, but here we used chemically induced proximity to convert kinase inhibitors into activators of therapeutic genes. We synthesized bivalent molecules that link ligands of the transcription factor B cell lymphoma 6 (BCL6) to inhibitors of cyclin-dependent kinases (CDKs). These molecules relocalized CDK9 to BCL6-bound DNA and directed phosphorylation of RNA polymerase II. The resulting expression of pro-apoptotic, BCL6-target genes caused killing of diffuse large B cell lymphoma cells and specific ablation of the BCL6-regulated germinal center response. Genomics and proteomics corroborated a gain-of-function mechanism in which global kinase activity was not inhibited but rather redirected. Thus, kinase inhibitors can be used to context-specifically activate transcription.

RIPK4 promotes oxidative stress and ferroptotic death through the downregulation of ACSM1

One of the most critical axes for cell fate determination is how cells respond to excessive reactive oxygen species (ROS)-oxidative stress. Extensive lipid peroxidation commits cells to death via a distinct cell death paradigm termed ferroptosis. However, the molecular mechanism regulating cellular fates to distinct ROS remains incompletely understood. Through siRNA against human receptor-interacting protein kinase (RIPK) family members, we found that RIPK4 is crucial for oxidative stress and ferroptotic death. Upon ROS induction, RIPK4 is rapidly activated, and the kinase activity of RIPK4 is indispensable to induce cell death. Specific ablation of RIPK4 in kidney proximal tubules protects mice from acute kidney injury induced by cisplatin and renal ischemia/reperfusion. RNA sequencing revealed the dramatically decreased expression of acyl-CoA synthetase medium-chain (ACSM) family members induced by cisplatin treatment which is compromised in RIPK4-deficient mice. Among these ACSM family members, suppression of ACSM1 strongly augments oxidative stress and ferroptotic cell death with induced expression of ACS long-chain family member 4, an important component for ferroptosis execution. Our lipidome analysis revealed that overexpression of ACSM1 leads to the accumulation of monounsaturated fatty acids, attenuation of polyunsaturated fatty acid (PUFAs) production, and thereby cellular resistance to ferroptosis. Hence, knockdown of ACSM1 resensitizes RIPK4 KO cells to oxidative stress and ferroptotic death. In conclusion, RIPK4 is a key player involved in oxidative stress and ferroptotic death, which is potentially important for a broad spectrum of human pathologies. The link between the RIPK4-ASCM1 axis to PUFAs and ferroptosis reveals a unique mechanism to oxidative stress-induced necrosis and ferroptosis.

Anatomical Ablation of the Atrioventricular Node.

Atrioventricular (AV) conduction ablation has been achieved by targeting the area of penetration of the conduction axis as defined by recording a His bundle potential. Ablation of the His bundle may reduce the possibility of a robust junctional escape rhythm. It was hypothesised that specific AV nodal ablation is feasible and safe. The anatomical position of the AV node in relation to the site of penetration of the conduction axis was identified as described in dissections and histological sections of human hearts. Radiofrequency (RF) ablation was accomplished based on the anatomical criteria. Specific anatomical ablation of the AV node was attempted in 72 patients. Successful AV nodal ablation was accomplished in 63 patients (87.5%), following 60 minutes (IQR 50-70 minutes) of procedure time, 3.4 minutes (IQR 2.4-5.5 minutes) of fluoroscopy time, and delivery of 4 (IQR 3-6) RF lesions. An escape rhythm was present in 45 patients (71%), and the QRS complex was similar to that before ablation in all 45 patients. Atropine was administered in six patients after the 10-min waiting period and did not result in restoration of conduction. In nine patients, AV conduction could not be interrupted, and AV block was achieved with ablation of the His after delivery of 12 (IQR 8-15) RF lesions. No cases of sudden death were encountered, and all patients had persistent AV block during a median 10.5 months (IQR 5-14 months) of follow-up. Anatomical ablation of the AV node is feasible and safe, and results in an escape rhythm similar to that before ablation.

Spliceosomal GTPase Eftud2 deficiency-triggered ferroptosis leads to Purkinje cell degeneration

Spliceosomal GTPase elongation factor Tu GTP binding domain containing 2 (EFTUD2) is a causative gene for mandibulofacial dysostosis with microcephaly (MFDM) syndrome comprising cerebellar hypoplasia and motor dysfunction. How EFTUD2 deficiency contributes to these symptoms remains elusive. Here, we demonstrate that specific ablation of Eftud2 in cerebellar Purkinje cells (PCs) in mice results in severe ferroptosis, PC degeneration, dyskinesia, and cerebellar atrophy, which recapitulates phenotypes observed in patients with MFDM. Mechanistically, Eftud2 promotes Scd1 and Gch1 expression, upregulates monounsaturated fatty acid phospholipids, and enhances antioxidant activity, thereby suppressing PC ferroptosis. Importantly, we identified transcription factor Atf4 as a downstream target to regulate anti-ferroptosis effects in PCs in a p53-independent manner. Inhibiting ferroptosis efficiently rescued cerebellar deficits in Eftud2 cKO mice. Our data reveal an important role of Eftud2 in maintaining PC survival, showing that pharmacologically or genetically inhibiting ferroptosis may be a promising therapeutic strategy for EFTUD2 deficiency-induced disorders.

Kir6.1, a component of an ATP-sensitive potassium channel, regulates natural killer cell development.

Involved in immunity and reproduction, natural killer (NK) cells offer opportunities to develop new immunotherapies to treat infections and cancer or to alleviate pregnancy complications. Most current strategies use cytokines or antibodies to enhance NK-cell function, but none use ion channel modulators, which are widely used in clinical practice to treat hypertension, diabetes, epilepsy, and other conditions. Little is known about ion channels in NK cells. We show that Kcnj8, which codes for the Kir6.1 subunit of a certain type of ATP-sensitive potassium (K ATP ) channel, is highly expressed in murine splenic and uterine NK cells compared to other K + channels previously identified in NK cells. Kcnj8 expression is highest in the most mature subset of splenic NK cells (CD27 - CD11b + ) and in NKG2A + or Ly49C/I + educated uterine NK cells. Using patch clamping, we show that a subset of NK cells expresses a current sensitive to the Kir6.1 blocker PNU-37883A. Kcnj8 does not participate in NK cell degranulation in response to tumor cells in vitro or rejection of tumor cells in vivo . Transcriptomics show that genes previously implicated in NK cell development are amongst those differentially expressed in CD27 - CD11b + NK cells deficient of Kcnj8 . Indeed, we found that mice with NK-cell specific Kcnj8 gene ablation have fewer CD11b + CD27 - and KLRG-1 + NK cells in the bone barrow and spleen. These results show that the K ATP subunit Kir6.1 has a key role in NK-cell development.

Centre-Specific Variation in Atrial Fibrillation Ablation-Treatment Rates in a Universal Single-Payer Healthcare System

BackgroundDisparities in atrial fibrillation ablation rates have been studied previously, with a focus on either patient characteristics or systems factors, rather than geographic factors. The impact of electrophysiology (EP) centre practice patterns on ablation rates has not been well studied. MethodsThis population-based cohort study used linked administrative datasets covering physician billing codes, hospitalizations, prescriptions, and census data. The study population consisted of patients who visited an emergency department with a new diagnosis of atrial fibrillation, in the period 2007-2016, in Ontario, Canada. Patient characteristics, including age, sex, medical history, comorbidities, socioeconomic factors, closest EP centre within 20 km, and distance to the nearest centre, were used as predictors in multivariable logistic regression models to assess the relationship between living in a location around specific EP centres and ablation rates. ResultsThe cohort included 134,820 patients, of whom 9267 had an ablation treatment during the study period. Patients undergoing ablation treatment were younger, had a lower Congestive Heart Failure, Hypertension, Age, Diabetes, Stroke/Transient Ischemic Attack (CHADS2) score, lived closer to EP centres, and had fewer comorbidities than those who did not receive ablation treatment. Wide variation occurred in ablation rates, with adjacent census divisions having ablation rates up to 2.6 times higher. Multivariate regression revealed significant differences in ablation rates for patients who lived in a location around certain EP centres. The odds ratios for living in a location closest to specific centres ranged from 0.78 (95% confidence interval: 0.68-0.89) to 1.60 (95% confidence interval:1.34-1.90). ConclusionsLiving near specific EP centres may significantly affect a patient’s likelihood of receiving ablation treatment, regardless of factors such as age, gender, socioeconomic status, prior medical history, and distance to EP centres.

Adenosine triggers early astrocyte reactivity that provokes microglial responses and drives the pathogenesis of sepsis-associated encephalopathy in mice

Molecular pathways mediating systemic inflammation entering the brain parenchyma to induce sepsis-associated encephalopathy (SAE) remain elusive. Here, we report that in mice during the first 6 hours of peripheral lipopolysaccharide (LPS)-evoked systemic inflammation (6 hpi), the plasma level of adenosine quickly increased and enhanced the tone of central extracellular adenosine which then provoked neuroinflammation by triggering early astrocyte reactivity. Specific ablation of astrocytic Gi protein-coupled A1 adenosine receptors (A1ARs) prevented this early reactivity and reduced the levels of inflammatory factors (e.g., CCL2, CCL5, and CXCL1) in astrocytes, thereby alleviating microglial reaction, ameliorating blood-brain barrier disruption, peripheral immune cell infiltration, neuronal dysfunction, and depression-like behaviour in the mice. Chemogenetic stimulation of Gi signaling in A1AR-deficent astrocytes at 2 and 4 hpi of LPS injection could restore neuroinflammation and depression-like behaviour, highlighting astrocytes rather than microglia as early drivers of neuroinflammation. Our results identify early astrocyte reactivity towards peripheral and central levels of adenosine as an important pathway driving SAE and highlight the potential of targeting A1ARs for therapeutic intervention.

Lipid Nanoparticle-mRNA Engineered Dendritic Cell Based Adoptive Cell Therapy Enhances Cancer Immune Response.

Lipid nanoparticles encapsulating mRNA (LNP-mRNA) revolutionized medicine over the past several years. While clinically approved indications currently focus on infectious disease vaccination, LNP-mRNA based treatments also hold promise for cancer immunotherapy. However, the route of dosing may impact treatment efficacy, safety, and dose. To minimize adverse effects, it is hypothesized that LNP-mRNA can be used to activate and engineer dendritic cells (DC) ex vivo before re-administration of these cells. Here, it is shown that LNP-mRNA engineered DCs can indeed vaccinate recipient mice. Vaccinated mice showed strong anti-tumor T cell responses, rejected tumor challenge, and displayed no evidence of toxicity. Further, it is found that DC specific ablation of the immune activating kinase NFkB inducing kinase (NIK) abrogated vaccination efficacy, demonstrating that adoptively transferred DCs can be functionally modified in addition to their antigen presentation capacity. Collectively, these studies show that ex vivo LNP-mRNA engineering of DCs is a feasible and robust therapeutic strategy for cancer.

Abstract PO-019: Ubiquitin E3 ligase OSTM1 regulates the cAMP/PKA/CREB pathway and suppresses B-cell malignancies

Abstract B-cell malignancies (BCM) often arise from genomic alterations introduced during different stages of B-cell development. Here, we performed a whole genome targeting CRISPR/Cas9 screen in the immortalized yet non-transformed IL-3-dependent Ba/F3 murine pro-B cell line to identify genes whose loss confers IL-3 independence, reasoning that these genes may function as tumor suppressor genes (TSGs) in BCM. One of the candidate genes is osteoporosis-associated transmembrane protein 1 (OSTM1), an E3 ubiquitin ligase whose loss-of-function causes autosomal recessive osteoporosis. Informatics analysis of patient data showed that OSTM1 is frequently deleted across BCM, which co-occurs with the deletion of a well-characterized TSG Cdkn2a. Genetic silencing of OSTM1 in Ba/F3 cells conferred IL-3-independent survival and proliferation, as well as splenomegaly, lymphadenopathy, and abnormal peripheral blood cell count, indicative of transformation in vivo. Using mass spectrometry screens, we identified phosphodiesterase 3B (PDE3B) as an interacting partner of OSTM1. OSTM1 ubiquitylates PDE3B and promotes its proteasomal degradation. The OSTM1-PDE3B interaction and PDE3B degradation are negatively regulated by OSTM1 N-glycosylation. As PDE3B catalyzes the conversion of cAMP to AMP, it negatively regulates the cAMP-dependent PKA/CREB/CREBBP tumor suppressive pathway. Indeed, overexpression of PDE3B accelerated cell proliferation while PDE3B silencing in OSTM1 KO cells reversed the transformation phenotype in cell lines and in mouse allograft tumors. Importantly, a strong correlation between OSTM1 disruption, increased PDE3B stability, and decreased PKA/CREB/CEBBP signaling was observed in cell lines and in BCM patient datasets. Finally, CD19-Cre mediated B-cell specific ablation of OSTM1 cooperated with CDKN2A deletion to drive spontaneous immature B-cell lymphomagenesis in mice. Collectively, these results indicate that OSTM1 is a novel TSG which targets PDE3B for proteasomal degradation. Loss of OSTM1 enhances PDE3B stability and abrogates the tumor-suppressive role of cAMP/CREB/CREBBP pathway which promotes BCM. Citation Format: Muhammad Usama Tariq, Julia Shen, Jaeyong Jung, Namratha Sheshadri, Junrong Yan, Rongrong Li, Kevin Lu, Tong Liu, Hong Li, Yue Lynn Wang, Ping Xie, Wei-Xing Zong. Ubiquitin E3 ligase OSTM1 regulates the cAMP/PKA/CREB pathway and suppresses B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-019.

Abstract PO-035: Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies

Abstract Ba/F3 is a murine pro-B cell line that is dependent on interleukin-3 (IL-3) for its survival and proliferation. It has been widely used for identifying oncogenes and mechanisms of drug-resistance. Using this cell line, we conducted a genome-wide CRISPR knockout screen to identify genes whose loss can confer IL-3 independence, hence may be implied as tumor suppressor genes (TSGs). We identified several genes that map to human chromosome 6q, a frequent deletion hotspot in B-cell malignancies, that may act as TSGs. One of these putative novel TSGs is Slc35a1, which encodes the membrane solute carrier that facilitates the transport of nucleotide sugar (CMP-sialic acid) into the Golgi apparatus for subsequent sialylation. Interestingly, in addition to Slc35a1, pathway analysis of hits from the CRISPR screen identified several genes in the sialic acid metabolism pathway, Nans, Cmas, and St3gal5. We confirmed that their silencing led to decreased protein sialyation and could confer IL-3-independent cell survival and growth in Ba/F3 cells. We continued to characterize Slc35a1 since its homozygous and heterozygous deletions are seen in 7-40% of different types of B-cell malignancies, including approximately 40% of patients with Diffuse Large B-cell Lymphoma. Slc35a1 deletion in murine and human cell-lines conferred cytokine-independent growth. Tail-vein injection of Slc35a1-silenced Ba/F3 cells resulted in allograft tumor growth presenting with splenomegaly and lymphadenopathy. Using Maackia amurensis lectin II (MAL II) to pull down sialylated proteins for mass spectrometry, we identified proteins that were differentially sialylated in Slc35a1 wild-type and knockout cells, among which are the α and β subunits of the integrin receptor, lymphocyte functional antigen 1 (LFA-1). As integrin desialylation can promote its constitutive activation, we are currently studying the impact of LFA-1 sialylation on B-cell function and transformation. We have also developed mouse models with B-cell specific ablation of Nans and Slc35a1 to further evaluate the role of sialylation on B-cell development and tumorigenesis in vivo. Overall, our study demonstrates that sialylation may function as a tumor suppressive mechanism and serve as a therapeutic target in B-cell cancers. Citation Format: Namratha Sheshadri, Rongrong Li, Muhammad Usama Tariq, Jianliang Shen, Jaeyong Jung, Junrong Yan, Kevin Lu, Zhiyuan Shen, Ping Xie, Wei-Xing Zong. Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-035.

Cell Ablation Techniques for the Larval Drosophila Neuromuscular System.

Tissue development requires local and long-distance communication between cells. Cell ablation experiments have provided critical insights into the functions of specific cell types and the tissue surrounding the dead cells. In the Drosophila neuromuscular system, ablation of motor neurons and muscles has revealed the roles of the ablated cells in axon pathfinding and circuit wiring. For example, when muscles are denervated due to laser ablation of their motor neuron inputs, they receive ectopic innervation from neighboring motor neurons. Here, we describe two methods of specific cell ablation. The first is a genetic ablation approach that uses GAL4 (ideally expressed in a small subset of cells) to drive expression of cell death genes reaper and head involution defective The second method relies on reactive oxygen species produced by light activation of the Arabidopsis-derived Singlet Oxygen Generator, miniSOG2, expressed in a subset of cells. For the latter, the precision stems from both the GAL4 and the restricting of the blue-light stimulation area.

More than the clock: distinct regulation of muscle function and metabolism by PER2 and RORα

AbstractCircadian rhythms, governed by the dominant central clock, in addition to various peripheral clocks, regulate almost all biological processes, including sleep–wake cycles, hormone secretion and metabolism. In certain contexts, the regulation and function of the peripheral oscillations can be decoupled from the central clock. However, the specific mechanisms underlying muscle‐intrinsic clock‐dependent modulation of muscle function and metabolism remain unclear. We investigated the outcome of perturbations of the primary and secondary feedback loops of the molecular clock in skeletal muscle by specific gene ablation of Period circadian regulator 2 (Per2) and RAR‐related orphan receptor alpha (Rorα), respectively. In both models, a dampening of core clock gene oscillation was observed, while the phase was preserved. Moreover, both loops seem to be involved in the homeostasis of amine groups. Highly divergent outcomes were seen for overall muscle gene expression, primarily affecting circadian rhythmicity in the PER2 knockouts and non‐oscillating genes in the RORα knockouts, leading to distinct outcomes in terms of metabolome and phenotype. These results highlight the entanglement of the molecular clock and muscle plasticity and allude to specific functions of different clock components, i.e. the primary and secondary feedback loops, in this context. The reciprocal interaction between muscle contractility and circadian clocks might therefore be instrumental to determining a finely tuned adaptation of muscle tissue to perturbations in health and disease. imageKey points Specific perturbations of the primary and secondary feedback loop of the molecular clock result in specific outcomes on muscle metabolism and function. Ablation of Per2 (primary loop) or Rorα (secondary loop) blunts the amplitude of core clock genes, in absence of a shift in phase. Perturbation of the primary feedback loop by deletion of PER2 primarily affects muscle gene oscillation. Knockout of RORα and the ensuing modulation of the secondary loop results in the aberrant expression of a large number of non‐clock genes and proteins. The deletion of PER2 and RORα affects muscle metabolism and contractile function in a circadian manner, highlighting the central role of the molecular clock in modulating muscle plasticity.

A Chemically Inducible Muscle Ablation System for the Zebrafish.

An effective method for tissue-specific ablation in zebrafish is the nitroreductase (NTR)/metronidazole (MTZ) system. Expressing bacterial NTR in the presence of nitroimidazole compounds causes apoptotic cell death, which can be useful for understanding many biological processes. However, this requires tissue-specific expression of the NTR enzyme, and many tissues have yet to be targeted with transgenic lines that express NTR. We generated a transgenic zebrafish line expressing NTR in differentiated skeletal muscle. Treatment of embryos with MTZ caused muscle specific cell ablation. We demonstrate this line can be used to monitor muscle regeneration in whole embryos and in transplanted transgenic cells.

An attention-based bilateral feature fusion network for 3D point cloud.

The widespread use of deep learning in processing point cloud data promotes the development of neural networks designed for point clouds. Point-based methods are increasingly becoming the mainstream in point cloud neural networks due to their high efficiency and performance. However, most of these methods struggle to balance both the geometric and semantic space of the point cloud, which usually leads to unclear local feature aggregation in geometric space and poor global feature extraction in semantic space. To address these two defects, we propose a bilateral feature fusion module capable of combining geometric and semantic data from the point cloud to enhance local feature extraction. In addition, we propose an offset vector attention module for better extraction of global features from point clouds. We provide specific ablation studies and visualizations in the article to validate our key modules. Experimental results show that the proposed method performs superior in both point cloud classification and segmentation tasks.

Pulmonary vein isolation combined with a new protocol of linear ablation to treat persistent atrial fibrillation: a prospective cohort with 1 year follow-up

Abstract Background The clinical value of ablation strategies beyond pulmonary vein isolation (PVI) in patients with persistent atrial fibrillation (perFA) is not clearly established and the optimal strategy to cure perFA remains a challenge due to the high recurrence rate after PVI alone (up to 50% of patients during 1-year follow-up). Purpose To determine the additional therapeutic value of a new design of linear ablation in the left atrium (LA) added to PVI in patients with perFA. Methods In this prospective study consecutive patients with perFA were included and underwent PVI combined with a predefined homogeneous protocol of linear ablation in the LA: anterolateral line from the mitral annulus to the left superior pulmonary vein around the left atrial appendage, anteroseptal line and posterior wall isolation. Patients were followed during 12 months after ablation with daily 30 s single-lead ECG obtained with mobile applications. The main endpoint was the incidence of recurrences, defined as any documented, sustained (&amp;gt;30 s) episodes of atrial arrhythmia that occurred after a 3 months blanking period. Antiarrhythmic drugs were avoided after the blanking period. Results 59 patients were included (61 [IQR 53-69] years old, 76% male sex). Acutely successful PVI was achieved in all patients and the linear ablation protocol was performed in 52 (89,7%) patients. The linea ablation protocol was not completed in 6 patients due to the following reasons: repetitive AF triggering from the same PV in 2 patients, induction and successful focal ablation of left atrial flutter in 1 patient, respiratory failure in 1 patient with severe obstructive lung disease and excessively prolonged procedure time in 2 patients. 58 patients completed the 12 months follow-up period and 1 patient was lost due to end-stage heart failure. The survival free of atrial arrhythmia recurrences was 69.1% [95%CI 55.1-79.5] (Figure). No patient had recurrences of perAF (&amp;gt;7 days continuous AF). 9 (53%) patients had at least 1 episode of self-limited paroxysmal AF. In these patients the average paroxysmal AF burden during the follow-up was 24 hours/year [IQR 9-24 hours/year]. In another 8 (47%) patients the recurrent arrhythmia was atypical left atrial flutter exclusively The incidence of procedure-related complications was 6,9%: 1 pericardial bleeding, 1 acute pulmonary edema, 1 splenic infarction and 1 stroke); no patient died during the study period. Conclusions A combined strategy of PVI plus the specific linear ablation protocol used in this study is associated with a low rate of atrial arrhythmia recurrences and total absence of persistent AF after ablation. A randomized multicenter clinical trial is ongoing to validate these results.Ablation strategy.Kaplan-Meier curve: recurrences

ZebraReg-a novel platform for discovering regulators of cardiac regeneration using zebrafish.

Cardiovascular disease is the leading cause of death worldwide with myocardial infarction being the most prevalent. Currently, no cure is available to either prevent or revert the massive death of cardiomyocytes that occurs after a myocardial infarction. Adult mammalian hearts display a limited regeneration capacity, but it is insufficient to allow complete myocardial recovery. In contrast, the injured zebrafish heart muscle regenerates efficiently through robust proliferation of pre-existing myocardial cells. Thus, zebrafish allows its exploitation for studying the genetic programs behind cardiac regeneration, which may be present, albeit dormant, in the adult human heart. To this end, we have established ZebraReg, a novel and versatile automated platform for studying heart regeneration kinetics after the specific ablation of cardiomyocytes in zebrafish larvae. In combination with automated heart imaging, the platform can be integrated with genetic or pharmacological approaches and used for medium-throughput screening of presumed modulators of heart regeneration. We demonstrate the versatility of the platform by identifying both anti- and pro-regenerative effects of genes and drugs. In conclusion, we present a tool which may be utilised to streamline the process of target validation of novel gene regulators of regeneration, and the discovery of new drug therapies to regenerate the heart after myocardial infarction.

TRPV1+ Sensory nerves modulate antigen specific immune responses

Abstract Sensory neurons recognize harmful or noxious stimuli and initiate a protective response by eliciting pain and defensive behavior. Nociceptors also interact with immune cells to regulate immune responses and play a key role in modulating antibody class switching to IgE. However, it is still unknown whether nociceptors participate in regulating primary IgG antibody responses to novel antigens. Here, we show that TRPV1 expressing nociceptors are required to develop an antigen specific immune response and that direct activation of TRPV1+ signals by selective optogenetic stimulation enhances primary IgG antibody responses to novel antigen. Specific ablation of TRPV1+ neurons, which include nociceptors, significantly blunts NP-specific IgG antibody responses in TRPV1-DTA mice compared to controls (anti-NP2 IgG AU, Day 28 control, 18,401 ± 3393 n=18 versus TRPV1-DTA, 9,028 ± 1,498, n=18, p&amp;lt; 0.001). Selective optogenetic stimulation of TRPV1+ nociceptors on the dorsum of the paw with blue light (473nm, 3Hz, 20%DC, 15min) immediately prior to a local immunization in TRPV1-ChR2 mice significantly increases KLH-specific IgG antibody responses compared to sham stimulation controls (anti-KLH IgG ng/mL Day 28, TRPV1-ChR2: 2,581 ± 476, n= 6 versus Control, 1,224 ± 332, n=8; * p&amp;lt;0.05). Together these studies with genetic and selective functional evidence indicates that nociceptors are required during immunization to produce primary antigen-specific IgG antibody responses to novel antigens.