Four-year follow-up of outcomes of the hybrid epicardial-endocardial ablation: A single-center experience.

BACKGROUND: The CONVERGE trial demonstrated that hybrid epicardial and endocardial ablation was more effective than catheter ablation for the treatment of persistent atrial fibrillation (AF) at 1 year. Long-term real-world outcome data are scarce. OBJECTIVE: We described a single-center experience by evaluating the long-term effectiveness and safety of hybrid epicardial-endocardial ablation. METHODS: This is a retrospective single-center study. Patients were followed up to 4 years. The primary end point was the rate of AF recurrence up to 4 years postablation. Secondary end points included reduction in antiarrhythmic therapy use, the effect of the ligament of Marshall removal, epicardial posterior wall, 3-dimensional mapping during epicardial ablation, and left atrial appendage exclusion as adjunct intraoperative interventions for AF recurrence. RESULTS: Of the 170 patients, 86.5% had persistent AF and 13.5% had long-standing persistent AF. AF-free survival was 87.6% at 1 year, 76.9% at 2 years, 70.4% at 3 years, and 59.3% at 4 years. Antiarrhythmic drug use was 87.6% at baseline and reduced to 21%, 20.6%, 18%, and 14.1% at year 1, 2, 3, and 4, respectively (P < .01 for all). Three-dimensional epicardial mapping showed a significant reduction in combined recurrence from 42% to 25% over 4 years of follow-up (P = .023). Ligament of Marshall and left atrial appendage exclusion showed numerical reduction in AF recurrence from 35% to 26% (P = .49) and from 44% to 30% (P = .07). CONCLUSION: The hybrid convergent procedure reduces AF recurrence and the need for antiarrhythmic drugs and, while maintaining a good safety profile, for the treatment of persistent and long-standing persistent AF.

Network switches and their role in circadian clocks.

Circadian rhythms are generated by complex interactions among genes and proteins. Self-sustained ∼24 h oscillations require negative feedback loops and sufficiently strong nonlinearities that are the product of molecular and network switches. Here, we review common mechanisms to obtain switch-like behavior, including cooperativity, antagonistic enzymes, multisite phosphorylation, positive feedback, and sequestration. We discuss how network switches play a crucial role as essential components in cellular circadian clocks, serving as integral parts of transcription-translation feedback loops that form the basis of circadian rhythm generation. The design principles of network switches and circadian clocks are illustrated by representative mathematical models that include bistable systems and negative feedback loops combined with Hill functions. This work underscores the importance of negative feedback loops and network switches as essential design principles for biological oscillations, emphasizing how an understanding of theoretical concepts can provide insights into the mechanisms generating biological rhythms.

Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum.

The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

Transcription activator WCC recruits deacetylase HDA3 to control transcription dynamics and bursting in Neurospora.

RNA polymerase II initiates transcription either randomly or in bursts. We examined the light-dependent transcriptional activator White Collar Complex (WCC) of Neurospora to characterize the transcriptional dynamics of the strong vivid (vvd) promoter and the weaker frequency (frq) promoter. We show that WCC is not only an activator but also represses transcription by recruiting histone deacetylase 3 (HDA3). Our data suggest that bursts of frq transcription are governed by a long-lived refractory state established and maintained by WCC and HDA3 at the core promoter, whereas transcription of vvd is determined by WCC binding dynamics at an upstream activating sequence. Thus, in addition to stochastic binding of transcription factors, transcription factor-mediated repression may also influence transcriptional bursting.

Antisense Transcription of the Neurospora Frequency Gene Is Rhythmically Regulated by CSP-1 Repressor but Dispensable for Clock Function.

The circadian clock of Neurospora crassa is based on a negative transcriptional/translational feedback loops. The frequency (frq) gene controls the morning-specific rhythmic transcription of a sense RNA encoding FRQ, the negative element of the core circadian feedback loop. In addition, a long noncoding antisense RNA, qrf, is rhythmically transcribed in an evening-specific manner. It has been reported that the qrf rhythm relies on transcriptional interference with frq transcription and that complete suppression of qrf transcription impairs the circadian clock. We show here that qrf transcription is dispensable for circadian clock function. Rather, the evening-specific transcriptional rhythm of qrf is mediated by the morning-specific repressor CSP-1. Since CSP-1 expression is induced by light and glucose, this suggests a rhythmic coordination of qrf transcription with metabolism. However, a possible physiological significance for the circadian clock remains unclear, as suitable assays are not available.

Adaptation to glucose starvation is associated with molecular reorganization of the circadian clock in Neurospora crassa.

The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress.

Data-driven modelling captures dynamics of the circadian clock of Neurospora crassa.

Eukaryotic circadian clocks are based on self-sustaining, cell-autonomous oscillatory feedback loops that can synchronize with the environment via recurrent stimuli (zeitgebers) such as light. The components of biological clocks and their network interactions are becoming increasingly known, calling for a quantitative understanding of their role for clock function. However, the development of data-driven mathematical clock models has remained limited by the lack of sufficiently accurate data. Here we present a comprehensive model of the circadian clock of Neurospora crassa that describe free-running oscillations in constant darkness and entrainment in light-dark cycles. To parameterize the model, we measured high-resolution time courses of luciferase reporters of morning and evening specific clock genes in WT and a mutant strain. Fitting the model to such comprehensive data allowed estimating parameters governing circadian phase, period length and amplitude, and the response of genes to light cues. Our model suggests that functional maturation of the core clock protein Frequency causes a delay in negative feedback that is critical for generating circadian rhythms.

US for Traumatic Nerve Injury, Entrapment Neuropathy, and Imaging-guided Perineural Injection.

US is commonly performed to help diagnose traumatic peripheral nerve injury and entrapment neuropathy, particularly with superficial nerves, where higher spatial resolution provides an advantage over MRI. Other advantages of US include dynamic evaluation, easy contralateral comparison, fewer implant contraindications, less artifact from ferromagnetic debris, and facile needle guidance for perineural injections. The authors review peripheral nerve US for traumatic peripheral nerve injury with an emphasis on injury grading and entrapment neuropathy and describe best-practice techniques for US-guided perineural injections while highlighting specific techniques and indications. Online supplemental material is available for this article. ©RSNA, 2022.

How circadian clocks keep time: the discovery of slowness.

Circadian clocks are molecular timers that measure 24-hour periods through a series of slow but precise biochemical processes. While circadian clocks of cyanobacteria are driven by interactions and ordered phosphorylation of highly structured protein assemblies, timekeeping by circadian clocks of eukaryotes involves slow progressive hyperphosphorylation of a large number of redundant sites in intrinsically disordered regions of clock proteins.

Neurospora casein kinase 1a recruits the circadian clock protein FRQ via the C-terminal lobe of its kinase domain.

Timing by the circadian clock of Neurospora is associated with hyperphosphorylation of frequency (FRQ), which depends on anchoring casein kinase 1a (CK1a) to FRQ. It is not known how CK1a is anchored so that approximately 100 sites in FRQ can be targeted. Here, we identified two regions in CK1a, p1 and p2, that are required for anchoring to FRQ. Mutation of p1 or p2 impairs progressive hyperphosphorylation of FRQ. A p1-mutated strain is viable but its circadian clock is non-functional, whereas a p2-mutated strain is non-viable. Our data suggest that p1 and potentially also p2 in CK1a provide an interface for interaction with FRQ. Anchoring via p1-p2 leaves the active site of CK1a accessible for phosphorylation of FRQ at multiple sites.

Oxytocin exerts harmful cardiac repolarization prolonging effects in drug-induced LQTS.

Background: Oxytocin is used therapeutically in psychiatric patients. Many of these also receive anti-depressant or anti-psychotic drugs causing acquired long-QT-syndrome (LQTS) by blocking HERG/IKr. We previously identified an oxytocin-induced QT-prolongation in LQT2 rabbits, indicating potential harmful effects of combined therapy. We thus aimed to analyze the effects of dual therapy with oxytocin and fluoxetine/risperidone on cardiac repolarization. Methods: Effects of risperidone, fluoxetine and oxytocin on QT/QTc, short-term variability (STV) of QT, and APD were assessed in rabbits using in vivo ECG and ex vivo monophasic AP recordings in Langendorff-perfused hearts. Underlying mechanisms were assessed using patch clamp in isolated cardiomyocytes. Results: Oxytocin, fluoxetine and risperidone prolonged QTc and APD in whole hearts. The combination of fluoxetine + oxytocin resulted in further QTc- and APD-prolongation, risperidone + oxytocin tended to increase QTc and APD compared to monotherapy. Temporal QT instability, STVQTc was increased by oxytocin, fluoxetine / fluoxetine + oxytocin and risperidone / risperidone + oxytocin. Similar APD-prolonging effects were confirmed in isolated cardiomyocytes due to differential effects of the compounds on repolarizing ion currents: Oxytocin reduced IKs, fluoxetine and risperidone reduced IKr, resulting in additive effects on IKtotal-tail. In addition, oxytocin reduced IK1, further reducing the repolarization reserve. Conclusion: Oxytocin, risperidone and fluoxetine prolong QTc / APD. Combined treatment further prolongs QTc/APD due to differential effects on IKs and IK1 (block by oxytocin) and IKr (block by risperidone and fluoxetine), leading to pronounced impairment of repolarization reserve. Oxytocin should be used with caution in patients in the context of acquired LQTS.

Casein kinase 1 and disordered clock proteins form functionally equivalent, phospho-based circadian modules in fungi and mammals.

Circadian clocks are timing systems that rhythmically adjust physiology and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks are based on transcriptional-translational feedback loops (TTFLs). Yet TTFL-core components such as Frequency (FRQ) in Neurospora and Periods (PERs) in animals are not conserved, leaving unclear how a 24-h period is measured on the molecular level. Here, we show that CK1 is sufficient to promote FRQ and mouse PER2 (mPER2) hyperphosphorylation on a circadian timescale by targeting a large number of low-affinity phosphorylation sites. Slow phosphorylation kinetics rely on site-specific recruitment of Casein Kinase 1 (CK1) and access of intrinsically disordered segments of FRQ or mPER2 to bound CK1 and on CK1 autoinhibition. Compromising CK1 activity and substrate binding affects the circadian clock in Neurospora and mammalian cells, respectively. We propose that CK1 and the clock proteins FRQ and PERs form functionally equivalent, phospho-based timing modules in the core of the circadian clocks of fungi and animals.

Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

AIM: Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. METHODS AND RESULTS: We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2-5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and ß-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2-5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. CONCLUSIONS: Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or ß-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

Transgenic rabbit models for cardiac disease research.

To study the pathophysiology of human cardiac diseases and to develop novel treatment strategies, complex interactions of cardiac cells on cellular, tissue and on level of the whole heart need to be considered. As in vitro cell-based models do not depict the complexity of the human heart, animal models are used to obtain insights that can be translated to human diseases. Mice are the most commonly used animals in cardiac research. However, differences in electrophysiological and mechanical cardiac function and a different composition of electrical and contractile proteins limit the transferability of the knowledge gained. Moreover, the small heart size and fast heart rate are major disadvantages. In contrast to rodents, electrophysiological, mechanical and structural cardiac characteristics of rabbits resemble the human heart more closely, making them particularly suitable as an animal model for cardiac disease research. In this review, various methodological approaches for the generation of transgenic rabbits for cardiac disease research, such as pronuclear microinjection, the sleeping beauty transposon system and novel genome-editing methods (ZFN and CRISPR/Cas9)will be discussed. In the second section, we will introduce the different currently available transgenic rabbit models for monogenic cardiac diseases (such as long QT syndrome, short-QT syndrome and hypertrophic cardiomyopathy) in detail, especially in regard to their utility to increase the understanding of pathophysiological disease mechanisms and novel treatment options. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

A cost comparison of atrial fibrillation monitoring strategies after embolic stroke of undetermined source.

Background: Detection of atrial fibrillation (AF) in patients with embolic stroke of undetermined source (ESUS) is challenging due to its paroxysmal nature. We sought to assess AF detection with an insertable cardiac monitor (ICM) and to perform cost analysis for various AF monitoring strategies post-ESUS We applied this cost analysis modeling to recently published Stroke AF and Per Diem trials. Methods: Retrospective chart review was performed in consecutive hospitalized patients with ESUS who had ICM placed prior to discharge. Utilizing rate of ICM-detected AF and Medicare average payments, we modeled 30-day per-patient diagnostic costs of Immediate ICM insertion prior to discharge versus using a wearable monitor followed by ICM in patients with ESUS, from Medicare and patient out-of-pocket perspectives. Similar modeling strategy and cost analysis was applied to the Stroke AF and Per Diem trials. Results: In 192 ESUS patients, AF detection increased with length of monitoring: 7.3 % at 14 days, 9.4 % at 30 days, and 17.2 % after a median ~ 6 months (189 days). Cost modeling predicted that immediate ICM leads to $3683-$4070 lower Medicare payments per-patient and $1425-$1503 lower patient out-of-pocket costs compared to Wearable-to-ICM strategies. Using similar modeling in the PER DIEM and STROKE AF trials, the additive costs of the 30-day ELR to ICM strategy ranged from $3786-$3946 from a payer perspective and $1472-$1503 from a patient out-of-pocket perspective. Conclusions: Use of ICM immediately after ESUS is cost-saving compared to Wearable-to-ICM strategies, due to the cost and low diagnostic yield of short-term wearable cardiac monitoring.

Global Transcriptome Characterization and Assembly of the Thermophilic Ascomycete Chaetomium thermophilum.

A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome of Chaetomium thermophilum has been reported previously, but it is essentially limited to open reading frames (ORFs) of protein coding genes and contains only a few noncoding transcripts. In this study, we identified and annotated full-length transcripts of C. thermophilum by deep RNA sequencing. We annotated 7044 coding genes and 4567 noncoding genes. Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes as well as 2878 novel noncoding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help to understand the gene regulatory landscape in C. thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species.

MXD/MIZ1 transcription regulatory complexes activate the expression of MYC-repressed genes.

MXDs are transcription repressors that antagonize MYC-mediated gene activation. MYC, when associated with MIZ1, acts also as a repressor of a subset of genes, including p15 and p21. A role for MXDs in regulation of MYC-repressed genes is not known. We report that MXDs activate transcription of p15 and p21 in U2OS cells. This activation required DNA binding by MXDs and their interaction with MIZ1. MXD mutants deficient in MIZ1 binding interacted with the MYC-binding partner MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA-binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of target genes.

The circadian cryptochrome, CRY1, is a pro-tumorigenic factor that rhythmically modulates DNA repair.

Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.