Molecular Regulation of Circadian Chromatin.

Circadian rhythms are generated by transcriptional negative feedback loops and require histone modifications and chromatin remodeling to ensure appropriate timing and amplitude of clock gene expression. Circadian modifications to histones are important for transcriptional initiation and feedback inhibition serving as signaling platform for chromatin-remodeling enzymes. Current models indicate circadian-regulated facultative heterochromatin (CRFH) is a conserved mechanism at clock genes in Neurospora, Drosophila, and mice. CRFH consists of antiphasic rhythms in activating and repressive modifications generating chromatin states that cycle between transcriptionally permissive and nonpermissive. There are rhythms in histone H3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac) and histone H3 lysine 4 methylation (H3K4me) during activation; while deacetylation, histone H3 lysine 9 methylation (H3K9me) and heterochromatin protein 1 (HP1) are hallmarks of repression. ATP-dependent chromatin-remodeling enzymes control accessibility, nucleosome positioning/occupancy, and nuclear organization. In Neurospora, the rhythm in facultative heterochromatin is mediated by the frequency (frq) natural antisense transcript (NAT) qrf. While in mammals, histone deacetylases (HDACs), histone H3 lysine 9 methyltransferase (KMT1/SUV39), and components of nucleosome remodeling and deacetylase (NuRD) are part of the nuclear PERIOD complex (PER complex). Genomics efforts have found relationships among rhythmic chromatin modifications at clock-controlled genes (ccg) revealing circadian control of genome-wide chromatin states. There are also circadian clock-regulated lncRNAs with an emerging function that includes assisting in chromatin dynamics. In this review, we explore the connections between circadian clock, chromatin remodeling, lncRNAs, and CRFH and how these impact rhythmicity, amplitude, period, and phase of circadian clock genes.

A Model Incorporating Left Ventricular Impedance Index may be Explanatory for Late Pulmonary Vein Isolation Failure.

Objective: To study the influence of a flow-based Impedance Index to attempt to explain the persistent late failure rate of Pulmonary Vein Isolation (PVI) in patients with Atrial Fibrillation (AF). Background: We recently described a flow-based Impedance Index for left ventricular ejection into the aorta and noted an association with Major Adverse Cardiovascular Event Rate (MACE). While the Impedance Index is not routinely measured in PVI patients it approximates to measures derivable from the left ventricular ejection fraction (EF). We sought to assess the Impedance Index's influence on PVI failure rate in combination with indices of left atrial size. Methods: In AF patients (n=100) undergoing a Cardiovascular Magnetic Resonance (CMR) imaging examination prior to undergoing PVI we assessed baseline characteristics for their influence on the PVI failure rate at 3-12 months. Uni-variable and multi-variable binary logistic models were performed to find predictors of the PVI failure rate at follow-up. Results: All patients underwent PVI and CMR imaging. A total of 26 (26%) patients had late AF recurrence at 3-12 months follow-up. Multi-variable models that predicted PVI failure were: 1) the baseline Impedance Index and LA volume index (p<0.05) and 2) the baseline Impedance Index and the degree of mitral valve regurgitation (MR) (p<0.001). While the Impedance Index was derived from EF, EF per se was not a predictor of PVI failure (p=0.28). Conclusions: We have provided evidence of the influence of a flow-based Impedance Index on the PVI late failure rate which is significant and remains explanatory when adjusting for measures of atrial size, MR grade and LA volume index. Direct measure of the Impedance Index was not available here and was derived from EF measures. Further work is needed to directly measure the Impedance Index in a PVI population and determine the mechanism for the influence on PVI failure, which may lead to modification of the ablation procedure to improve the success rate.

Safety and efficacy of zero fluoroscopy transseptal puncture with different approaches.

INTRODUCTION: Atrial fibrillation (AF) ablation requires access to the left atrium (LA) via transseptal puncture (TP). TP is traditionally performed with fluoroscopic guidance. Use of intracardiac echocardiography (ICE) and three-dimensional mapping allows for zero fluoroscopy TP. OBJECTIVE: To demonstrate safety and efficacy of zero fluoroscopy TP using multiple procedural approaches. METHODS: Patients undergoing AF ablation between January 2015 and November 2017 at five institutions were included. ICE and three-dimensional mapping were used for sheath positioning and TP. Variable technical approaches were used across centers including placement of J wire in the superior vena cava with ICE guidance followed by dragging down the transseptal sheath into the interatrial septum, or guiding the transseptal sheath directly to the interatrial septum by localizing the ablation catheter with three-dimensional mapping and replacing it with the transseptal needle once in position. In patients with pacemaker/implantable cardiac defibrillator leads, pre-/poststudy device interrogation was performed. RESULTS: A total of 747 TPs were performed (646 patients, age 63.1 ± 13.1, 67.5% male, LA volume index 34.5 ± 15.8 mL/m2 , ejection fraction 57.7 ± 10.9%) with 100% success. No punctures required fluoroscopy. Two pericardial effusions, two pericardial tamponades requiring pericardiocentesis, and one transient ischemic attack were observed during the overall ablation procedure, with a total complication rate of 0.7%. There were no other periprocedural complications related to TP, including intrathoracic bleeding, stroke, or death both immediately following TP and within 30 days of the procedure. In patients with intracardiac devices, no device-related complications were observed. CONCLUSION: TP can be safely and effectively performed without the need for fluoroscopy.

BMAL1 associates with chromosome ends to control rhythms in TERRA and telomeric heterochromatin.

The circadian clock and aging are intertwined. Disruption to the normal diurnal rhythm accelerates aging and corresponds with telomere shortening. Telomere attrition also correlates with increase cellular senescence and incidence of chronic disease. In this report, we examined diurnal association of White Collar 2 (WC-2) in Neurospora and BMAL1 in zebrafish and mice and found that these circadian transcription factors associate with telomere DNA in a rhythmic fashion. We also identified a circadian rhythm in Telomeric Repeat-containing RNA (TERRA), a lncRNA transcribed from the telomere. The diurnal rhythm in TERRA was lost in the liver of Bmal1-/- mice indicating it is a circadian regulated transcript. There was also a BMAL1-dependent rhythm in H3K9me3 at the telomere in zebrafish brain and mouse liver, and this rhythm was lost with increasing age. Taken together, these results provide evidence that BMAL1 plays a direct role in telomere homeostasis by regulating rhythms in TERRA and heterochromatin. Loss of these rhythms may contribute to telomere erosion during aging.

Age modulates liver responses to asparaginase-induced amino acid stress in mice.

Asparaginase is an amino acid-depleting agent used to treat blood cancers. Metabolic complications due to asparaginase affect liver function in humans. To examine how the liver response to asparaginase changes during maturity to adulthood, here we treated juvenile (2-week), young adult (8-week), and mature adult (16-week) mice with drug or excipient for 1 week and conducted RNA-Seq and functional analyses. Asparaginase reduced body growth and liver mass in juveniles but not in the adult animals. Unbiased exploration of the effect of asparaginase on the liver transcriptome revealed that the integrated stress response (ISR) was the only molecular signature shared across the ages, corroborating similar eukaryotic initiation factor 2 phosphorylation responses to asparaginase at all ages. Juvenile livers exhibited steatosis and iron accumulation following asparaginase exposure along with a hepatic gene signature indicating that asparaginase uniquely affects lipid, cholesterol, and iron metabolism in juvenile mice. In contrast, asparaginase-treated adult mice displayed greater variability in liver function, which correlated with an acute-phase inflammatory response gene signature. Asparaginase-exposed adults also had a serine/glycine/one-carbon metabolism gene signature in liver that corresponded with reduced circulating glycine and serine levels. These results establish the ISR as a conserved response to asparaginase-mediated amino acid deprivation and provide new insights into the relationship between the liver transcriptome and hepatic function upon asparaginase exposure.

Histone H3 lysine 4 methyltransferase is required for facultative heterochromatin at specific loci.

BACKGROUND: Histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 lysine 9 tri-methylation (H3K9me3) are widely perceived to be opposing and often mutually exclusive chromatin modifications. However, both are needed for certain light-activated genes in Neurospora crassa (Neurospora), including frequency (frq) and vivid (vvd). Except for these 2 loci, little is known about how H3K4me3 and H3K9me3 impact and contribute to light-regulated gene expression. RESULTS: In this report, we performed a multi-dimensional genomic analysis to understand the role of H3K4me3 and H3K9me3 using the Neurospora light response as the system. RNA-seq on strains lacking H3 lysine 4 methyltransferase (KMT2/SET-1) and histone H3 lysine 9 methyltransferase (KMT1/DIM-5) revealed some light-activated genes had altered expression, but the light response was largely intact. Comparing these 2 mutants to wild-type (WT), we found that roughly equal numbers of genes showed elevated and reduced expression in the dark and the light making the environmental stimulus somewhat ancillary to the genome-wide effects. ChIP-seq experiments revealed H3K4me3 and H3K9me3 had only minor changes in response to light in WT, but there were notable alterations in H3K4me3 in Δkmt1/Δdim-5 and H3K9me3 in Δkmt2/Δset-1 indicating crosstalk and redistribution between the modifications. Integrated analysis of the RNA-seq and ChIP-seq highlighted context-dependent roles for KMT2/SET1 and KMT1/DIM-5 as either co-activators or co-repressors with some overlap as co-regulators. At a small subset of loci, H3K4 methylation is required for H3K9me3-mediated facultative heterochromatin including, the central clock gene frequency (frq). Finally, we used sequential ChIP (re-ChIP) experiment to confirm Neurospora contains K4/K9 bivalent domains. CONCLUSIONS: Collectively, these data indicate there are obfuscated regulatory roles for H3K4 methylation and H3K9 methylation depending on genome location with some minor overlap and co-dependency.

Nutrition Risk is Associated with Leukocyte Telomere Length in Middle-Aged Men and Women with at Least One Risk Factor for Cardiovascular Disease.

Poor diet quality has been associated with several age-related chronic conditions, but its relationship to telomere length, a biological marker of cellular aging, is unclear. The purpose of this cross-sectional study was to determine whether overall diet quality was associated with relative leukocyte telomere length (rLTL) in a sample (n = 96) of nonsmoking middle-aged adults in Appalachia with at least one risk factor for cardiovascular disease. Diet quality was assessed using the Healthy Eating Index (HEI-2015), the alternate Mediterranean diet score (aMed), and the Dietary Screening Tool (DST). Peripheral rLTL was measured by quantitative real-time polymerase chain reaction. The associations between potentially confounding sociodemographic, lifestyle and health-related factors and the first and fourth rLTL quartile groups were examined using Chi-square or Fisher's Exact tests or logistic regression. The relationships between diet quality index scores and rLTL as a continuous variable were analyzed using simple linear regression and multivariate linear models, analogous to linear covariance analyses. The rLTL ranged from 0.46 to 1.49 (mean ± SEM was 1.02 ± 0.18). Smoking history, income level, and cardiovascular health (Life's Simple 7) were associated with the lowest and highest quartiles of rLTL and were used as covariates. In adjusted and unadjusted models, participants considered "at nutrition risk" by the DST were more likely to have shorter rLTL than those "not at risk or at potential risk" (p = 0.004). However, there was no evidence that adherence to the 2015⁻2020 Dietary Guidelines for Americans or to a Mediterranean diet was associated with rLTL in this sample. Intervention studies are needed to determine if improving the diet quality of those at nutrition risk results in reduced telomere attrition over time.

Pulmonary vein remodeling following pulmonary vein isolation in patients with atrial fibrillation-do pulmonary veins represent only an epiphenomenon? A cardiac MRI study.

BACKGROUND: After successful pulmonary vein isolation (PVI) for atrial fibrillation (AF), the left atrium (LA) undergoes reverse remodeling. However, few studies have directly studied pulmonary vein (PV) remodeling and focused on whether pre PVI-PV conditions could predict outcome of the procedure. We hypothesize that: (I) post PVI, in addition to LA remodeling the PVs undergo a parallel degree of remodeling; and (II) that PV characteristics pre PVI can be used to identify patients more likely to sustain normal sinus rhythm (NSR). METHODS: Patients (n=100) scheduled for PVI had a cardiovascular magnetic resonance (CMR) imaging before and 6±2 months following PVI. PV cross sectional areas (CSA) within 0.5 cm of the ostium and LA volumes were measured. Patients were categorized as responders (R) or non-responders (NR), based on two separate 14-day Holter monitoring. RESULTS: PVs CSA were significantly reduced post procedure in both groups, R (233±53 to 192±52 mm2, P<0.001) and NR (241±54 to 207±44 mm2, P<0.001), however, the difference between R and NR post PVI was not significant (192±52 to 207±44 mm2, P=0.19). Reduction in PVs CSAs post procedure moderately correlated with the 3D LA volume reduction (r=0.48, P<0.001). CONCLUSIONS: PVs mirror the LA in that they significantly change in size following PVI yet they were not found to directly predict maintenance of NSR.

Long non-coding RNAs have age-dependent diurnal expression that coincides with age-related changes in genome-wide facultative heterochromatin.

BACKGROUND: Disrupted diurnal rhythms cause accelerated aging and an increased incidence in age-related disease and morbidity. The circadian clock governs cell physiology and metabolism by controlling transcription and chromatin. The goal of this study is to further understand the mechanism of age-related changes to circadian chromatin with a focus on facultative heterochromatin and diurnal non-coding RNAs. RESULTS: We performed a combined RNA-seq and ChIP-seq at two diurnal time-points for three different age groups to examine the connection between age-related changes to circadian transcription and heterochromatin in neuronal tissue. Our analysis focused on uncovering the relationships between long non-coding RNA (lncRNA) and age-related changes to histone H3 lysine 9 tri-methylation (H3K9me3), in part because the Period (Per) complex can direct facultative heterochromatin and models of aging suggest age-related changes to heterochromatin and DNA methylation. Our results reveal that lncRNAs and circadian output change dramatically with age, but the core clock genes remain rhythmic. Age-related changes in clock-controlled gene (ccg) expression indicate there are age-dependent circadian output that change from anabolic to catabolic processes during aging. In addition, there are diurnal and age-related changes in H3K9me3 that coincide with changes in transcription. CONCLUSIONS: The data suggest a model where some age-related changes in diurnal expression are partially attributed to age-related alterations to rhythmic facultative heterochromatin. The changes in heterochromatin are potentially mediated by changes in diurnal lncRNA creating an interlocked circadian-chromatin regulatory network that undergoes age-dependent metamorphosis.

Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase.

The anti-leukemic agent asparaginase activates the integrated stress response (ISR) kinase GCN2 and inhibits signaling via mechanistic target of rapamycin complex 1 (mTORC1). The study objective was to investigate the protective role of activating transcription factor 4 (ATF4) in controlling the hepatic transcriptome and mediating GCN2-mTORC1 signaling during asparaginase. We compared global gene expression patterns in livers from wildtype, Gcn2 -/-, and Atf4 -/- mice treated with asparaginase or excipient and further explored selected responses in livers from Atf4 +/- mice. Here, we show that ATF4 controls a hepatic gene expression profile that overlaps with GCN2 but is not required for downregulation of mTORC1 during asparaginase. Ingenuity pathway analysis indicates GCN2 independently influences inflammation-mediated hepatic processes whereas ATF4 uniquely associates with cholesterol metabolism and endoplasmic reticulum (ER) stress. Livers from Atf4 -/- or Atf4 +/- mice displayed an amplification of the amino acid response and ER stress response transcriptional signatures. In contrast, reduction in hepatic mTORC1 signaling was retained in Atf4 -/- mice treated with asparaginase. CONCLUSIONS: GCN2 and ATF4 serve complementary roles in the hepatic response to asparaginase. GCN2 functions to limit inflammation and mTORC1 signaling whereas ATF4 serves to limit the amino acid response and prevent ER stress during amino acid depletion by asparaginase.

The Arcuate Estrogen-Regulated Transcriptome: Estrogen Response Element-Dependent and -Independent Signaling of ERα in Female Mice.

To influence energy homeostasis and reproduction, 17ß-estradiol (E2) controls the arcuate nucleus (ARC) through multiple receptor-mediated mechanisms, but primarily via estrogen receptor (ER) α, which signals through both estrogen response element (ERE)-dependent and -independent mechanisms. To determine ERα-mediated, ERE-dependent, and ERE-independent E2 signaling in the ARC, we examined the differential regulation of the mouse arcuate transcriptome by E2 using three mice genotypes: (1) wild-type, (2) ERα knock-in/knockout (ERE-independent mechanisms), and (3) total ERα knockout (ERα-independent mechanisms). Females were ovariectomized and injected with oil or E2, and RNA sequencing on the ARC was used to identify E2-regulated genes in each genotype. Our results show that E2 regulates numerous genes involved in cell signaling, cytoskeleton structure, inflammation, neurotransmission, neuropeptide production, and transcription. Furthermore, ERE-independent signaling regulates ARC genes expressed in kisspeptin neurons and transcription factors that control the hypothalamic/pituitary/gonadal axis. Interestingly, a few genes involved in mitochondrial oxidative respiration were regulated by E2 through ERα-independent signaling. A comparison within oil- and E2-treated females across the three genotypes suggests that genes involved in cell growth and proliferation, extracellular matrices, neuropeptides, receptors, and transcription are differentially expressed across the genotypes. Comparing with previously published chromatin immunoprecipitation sequencing analysis, we found that ERE-independent regulation in the ARC is mainly mediated by tethering of ERα, which is consistent with previous findings. We conclude that the mouse arcuate estrogen-regulated transcriptome is regulated by multiple receptor-mediated mechanisms to modulate the central control of energy homeostasis and reproduction, including novel E2-responsive pathways.

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils.

Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.

The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin.

The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.

The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output.

Regulated DNA methylation and the circadian clock: implications in cancer.

Since the cloning and discovery of DNA methyltransferases (DNMT), there has been a growing interest in DNA methylation, its role as an epigenetic modification, how it is established and removed, along with the implications in development and disease. In recent years, it has become evident that dynamic DNA methylation accompanies the circadian clock and is found at clock genes in Neurospora, mice and cancer cells. The relationship among the circadian clock, cancer and DNA methylation at clock genes suggests a correlative indication that improper DNA methylation may influence clock gene expression, contributing to the etiology of cancer. The molecular mechanism underlying DNA methylation at clock loci is best studied in the filamentous fungi, Neurospora crassa, and recent data indicate a mechanism analogous to the RNA-dependent DNA methylation (RdDM) or RNAi-mediated facultative heterochromatin. Although it is still unclear, DNA methylation at clock genes may function as a terminal modification that serves to prevent the regulated removal of histone modifications. In this capacity, aberrant DNA methylation may serve as a readout of misregulated clock genes and not as the causative agent. This review explores the implications of DNA methylation at clock loci and describes what is currently known regarding the molecular mechanism underlying DNA methylation at circadian clock genes.

A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile.

Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2µm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.

Circadian rhythms synchronize mitosis in Neurospora crassa.

The cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. Alterations in this network may lead to diseases such as cancer. Therefore, it is critical to identify molecular components that connect these two oscillators. However, molecular mechanisms between the clock and the cell cycle remain largely unknown. A model filamentous fungus, Neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhythms, but not used to investigate the molecular coupling between these two oscillators. In this report, we show that a conserved coupling between the circadian clock and the cell cycle exists via serine/threonine protein kinase-29 (STK-29), the Neurospora homolog of mammalian WEE1 kinase. Based on this finding, we established a mathematical model that predicts circadian oscillations of cell cycle components and circadian clock-dependent synchronized nuclear divisions. We experimentally demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, oscillate in a circadian manner with bioluminescence reporters. The oscillations of clb-1 and stk-29 gene expression are abolished in a circadian arrhythmic frq(ko) mutant. Additionally, we show the light-induced phase shifts of a core circadian component, frq, as well as the gene expression of the cell cycle components clb-1 and stk-29, which may alter the timing of divisions. We then used a histone hH1-GFP reporter to observe nuclear divisions over time, and show that a large number of nuclear divisions occur in the evening. Our findings demonstrate the circadian clock-dependent molecular dynamics of cell cycle components that result in synchronized nuclear divisions in Neurospora.

Aging well with a little wine and a good clock.

Age-related decline in mammalian circadian rhythm has been recognized for decades, but the underlying molecular mechanisms have remained elusive. In this issue of Cell, Chang and Guarente use brain-specific SIRT1 knockout mice and transgenic mice overexpressing SIRT1 to develop an enticing model for how SIRT1 helps maintain the robustness of the aging circadian clock.

Mitral regurgitation recovery and atrial reverse remodeling following pulmonary vein isolation procedure in patients with atrial fibrillation: a clinical observation proof-of-concept cardiac MRI study.

BACKGROUND: Reverse remodeling of the left atrium (LA) following successful pulmonary vein isolation (PVI) in patients with atrial fibrillation (AF) has been well documented. However, mitral regurgitation (MR) recovery after successful PVI has never been demonstrated systematically. The objective of our study was to retrospectively analyze the effectiveness of PVI in patients with AF on recovery of MR using cardiac magnetic resonance (CMR) imaging. METHODS: Prior to PVI, patients underwent a clinically indicated CMR imaging. Post-PVI (6 ± 2 months), patients underwent a follow-up MRI and were classified into two groups-responders (R) and non-responders (NR) to PVI-as assessed by cessation of AF at the end of the prespecified 6-month (14-day "P" sensitive event monitor defined) follow-up period. Furthermore, CMR was used to evaluate the severity of MR (0 to 4+) and to relate changes in MR to LA volumes as well as mitral apparatus geometry. Patients who had mild and higher MR (2+) on baseline CMR and had a post-PVI CMR were selected for final analysis. RESULTS: Out of the consecutive 122 patients with AF who underwent PVI, 74 patients that had mitral regurgitation on initial CMR were included in the study. Of these74 patients with AF with MR, 52 (70 %) were classified as R and 22 (30 %) were classified as NR. Baseline demographics were similar between the groups. In the subgroup with mild to severe MR, pre vs. post in the R group MR severity significantly improved (mean = 2.3, median = 2.0 vs. mean = 1.0, median = 1.0, p < 0.0001) and was matched by favorable reverse remodeling of the mitral apparatus geometry (annulus = 35 ± 4 vs. 33 ± 3 mm, p < 0.002; tenting area = 175 ± 56 vs.137 ± 37 mm(2), p < 0.003; tenting height = 8 ± 2 vs.7 ± 2 mm, p < 0.02; and tenting angle = 129 ± 10° vs. 131 ± 11°, p = 0.1). However, in the NR subgroup, MR failed to improve (mean = 2.2, median = 2.0 vs. mean = 1.5, median = 1.0, p = NS) and paralleled general failure of mitral geometry reverse remodeling (annulus = 35 ± 4 vs. 35 ± 4 mm, p = 0.2; tenting area = 153 ± 39 vs. 152 ± 34 mm(2), p = NS; tenting height = 7 ± 1 vs. 7.0 ± 2, p = 0.1; and tenting angle = 131 ± 11° vs. 133 ± 10°, p = NS). In those with lesser degrees of MR, favorable remodeling was predicated on responder status to PVI. Similarly, other cardiac dimensions pre- to post-PVI favorably improved in the R group, but not in the NR group. CONCLUSION: In those with durable maintenance of normal sinus rhythm (NSR), cardiac reverse remodeling demonstrated by 3D CMR occurs and is matched by marked improvements in MR and mitral apparatus, likely contributing to continued maintenance of NSR.