The mammalian circadian clock uses interlocked negative feedback loops in which the heterodimeric basic helix-loop-helix transcription factor BMAL1/CLOCK is a master regulator. While there is ...prominent control of liver functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Using chromatin immunoprecipitation combined with deep sequencing we obtained a time-resolved and genome-wide map of BMAL1 binding in mouse liver, which allowed us to identify over 2,000 binding sites, with peak binding narrowly centered around Zeitgeber time 6. Annotation of BMAL1 targets confirms carbohydrate and lipid metabolism as the major output of the circadian clock in mouse liver. Moreover, transcription regulators are largely overrepresented, several of which also exhibit circadian activity. Genes of the core circadian oscillator stand out as strongly bound, often at promoter and distal sites. Genomic sequence analysis of the sites identified E-boxes and tandem E1-E2 consensus elements. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution centered at Zeitgeber time 10. Importantly, sites with E1-E2 elements showed tighter phases both in binding and mRNA accumulation. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and post-transcriptional regulation contribute differentially to circadian expression phase. Together, our analysis of a dynamic protein-DNA interactome uncovered how genes of the core circadian oscillator crosstalk and drive phase-specific circadian output programs in a complex tissue.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Circadian clocks are self-sustained oscillators that orchestrate metabolism and physiology in synchrony with the 24-h day–night cycle. They are temperature compensated over a wide range and entrained ...by daily recurring environmental cues. Eukaryotic circadian clocks are governed by cell-based transcriptional–translational feedback loops (TTFLs). The core components of the TTFLs are largely known and their molecular interactions in many cases well established. Although the core clock components are not or only partly conserved, the molecular wiring of TTFLs is rather similar across kingdoms and phylae. In all known systems, circadian timing relies critically on casein kinase 1 (CK1) and CK1-dependent hyperphosphorylation of core clock proteins, in particular of negative elements of the TTFLs. Yet, we lack concepts as to how phosphorylation by CK1a and other kinases relates to timekeeping on the molecular level. Here we summarize what is known about phosphorylation of core components of the circadian clock of Neurospora crassa and speculate about the molecular basis of circadian timekeeping by hyperphosphorylation of intrinsically disordered regions in clock proteins.
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•Phosphorylation and regulation of FRQ•Role of CK1a and other kinases•Phosphorylation and regulation of the WCC•Circadian versus light-induced phosphorylation•Hypothesis of molecular time measurement
It is difficult to predict adverse patient outcomes associated with transvenous lead extraction (TLE) procedures.
The purpose of this study was to examine the safety and efficacy of chronic ...endovascular pacemaker and implantable cardioverter-defibrillator (ICD) lead extraction and risk factors associated with adverse patient outcomes.
Consecutive patients undergoing TLE at the Cleveland Clinic between August 1996 and August 2011 were included in the analysis. Univariate and multivariable logistic regression analyses were performed to evaluate for associations with outcomes. Continuous data are given as median (25th, 75th percentile). Categorical data are given as number (percentage).
In total, 5521 leads (4137 74.9% pacemaker, 1384 25.1% ICD) were extracted during 2999 TLE procedures (patient age 67.2 55.2, 76.2 years, 30.2% female). Lead implant duration was 4.7 (2.4, 8.3) years, and 2.0 (1.0, 2.0) leads were extracted per procedure. Powered sheaths were used in 74.9% of procedures. Overall, there was 95.1% complete procedural success, 98.9% clinical success, and 1.1% failure, with 3.6% minor complications and 1.8% major complications. All-cause mortality within 30 days of TLE was 2.2%. Multivariable predictors of major complications included cerebrovascular disease, ejection fraction ≤15%, lower platelet count, international normalized ratio ≥1.2, mechanical sheaths, and powered sheaths. Multivariable predictors of all-cause mortality within 30 days of TLE included body mass index <25 kg/m(2), end-stage renal disease, higher New York Heart Association functional class, lower hemoglobin, higher international normalized ratio, lead extraction for infection, and extraction of a dual-coil ICD lead.
TLE in this single-center experience was highly successful. Risk factors associated with adverse patient outcomes were identified.
In a phase 1 trial, six healthy male volunteers received 0.1 mg per kilogram of body weight of a superagonistic anti-CD28 monoclonal antibody. Unexpectedly, all six volunteers had a transient ...critical illness characterized by multiorgan failure. These events give a view of a specific form of the cytokine-release syndrome in the absence of underlying medical disease.
Six volunteers who received the anti-CD28 monoclonal antibody had a transient critical illness characterized by multiorgan failure. These events give a view of a specific form of the cytokine-release syndrome in the absence of underlying medical disease.
On March 13, 2006, eight healthy male volunteers participated in a double-blind, randomized, placebo-controlled phase 1 study of the safety of TGN1412 (TeGenero), a novel monoclonal antibody. The study drug is a recombinantly expressed, humanized superagonist anti-CD28 monoclonal antibody of the IgG4κ subclass that stimulates and expands T cells independently of the ligation of the T-cell receptor.
1
In contrast to other antibodies in clinical use or in clinical trials, TGN1412 directly stimulates the immune response in vivo. In preclinical models, the stimulation of CD28 with TGN1412 (or with murine-antibody counterparts) preferentially activated and expanded type 2 helper T cells
2
and, . . .
The outcomes of patients requiring emergent surgical or endovascular intervention during transvenous lead extraction (TLE) have not been well characterized.
To evaluate the incidence of catastrophic ...complications requiring emergent surgical or endovascular intervention during TLE, to describe the injuries, and to review patient management and outcomes.
Consecutive patients undergoing TLE of pacemaker and implantable cardioverter-defibrillator (ICD) leads at the Cleveland Clinic between August 1996 and September 2012 were included in the analysis.
A total of 5973 (4436 74.3% pacemaker and 1537 25.7% ICD) leads were extracted during 3258 TLE procedures (median 25th, 75th percentile patient age 67.0 55.0, 76.1 years; 69.2% men). The median (25th, 75th percentile) lead implant duration was 4.9 (2.4, 8.4) years, and 2.0 (1.0, 2.0) leads were extracted per procedure. Powered sheaths were used in 2369 (72.7%) procedures. Twenty-five (0.8%) patients experienced catastrophic complications requiring emergent surgical or endovascular intervention. Twenty patients (0.6%) required either sternotomy (n = 18) or thoracotomy (n = 2) for superior vena cava laceration (n = 15) and right atrial (n = 2) or ventricular (n = 3) perforation. Two patients required vascular repair at the procedural access site for either subclavian vein or artery laceration. Three patients were managed with an endovascular approach for superior vena cava laceration, left axillary artery laceration, and brachiocephalic vein and artery fistula. In-hospital mortality was 36.0% (6 procedural/operative deaths and 3 deaths during the same hospitalization).
Major vascular injury or cardiac perforation requiring emergent surgical or endovascular intervention during TLE is uncommon but carries significant in-hospital mortality. Despite high mortality, nearly two-thirds of these patients were rescued with immediate response and surgical or endovascular intervention.
Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic ...recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological ELISA using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 reverse transcription PCR-confirmed, SARS-CoV-2-hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies against the 2 different viral proteins showed a moderate correlation. Antibodies against N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.9% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD-binding antibodies exhibited neutralizing capacity, but only 74% of N-protein-positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein-binding antibodies does not always correlate with presence of S-RBD-neutralizing antibodies and caution against the extensive use of N-protein-based serology testing for determination of potential COVID-19 immunity.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Autonomous endogenous time-keeping is ubiquitous across many living organisms, known as the circadian clock when it has a period of about 24 h. Interestingly, the fundamental design principle with a ...network of interconnected negative and positive feedback loops is conserved through evolution, although the molecular components differ. Filamentous fungus
is a well-established chrono-genetics model organism to investigate the underlying mechanisms. The core negative feedback loop of the clock of
is composed of the transcription activator White Collar Complex (WCC) (heterodimer of WC1 and WC2) and the inhibitory element called FFC complex, which is made of FRQ (Frequency protein), FRH (Frequency interacting RNA Helicase) and CK1a (Casein kinase 1a). While exploring their temporal dynamics, we investigate how limit cycle oscillations arise and how molecular switches support self-sustained rhythms. We develop a mathematical model of 10 variables with 26 parameters to understand the interactions and feedback among WC1 and FFC elements in nuclear and cytoplasmic compartments. We performed control and bifurcation analysis to show that our novel model produces robust oscillations with a wild-type period of 22.5 h. Our model reveals a switch between WC1-induced transcription and FFC-assisted inactivation of WC1. Using the new model, we also study the possible mechanisms of glucose compensation. A fairly simple model with just three nonlinearities helps to elucidate clock dynamics, revealing a mechanism of rhythms' production. The model can further be utilized to study entrainment and temperature compensation.
For each clinical circumstance, the benefits of transvenous lead extraction (TLE) need to be weighed against the risks. Clinical decision-making tools for predicting mortality after TLE are lacking.
...To create a preoperative risk score for prediction of 30-day all-cause mortality after TLE of pacemaker and defibrillator leads.
Consecutive patients undergoing TLE at the Cleveland Clinic between August 1996 and August 2011 were included in the analysis. A risk nomogram for predicting 30-day all-cause mortality was developed using baseline clinical variables and multivariable logistic regression modeling. Discrimination and calibration were assessed by using bootstrapping for internal validation. Continuous data are presented as median (25th, 75th percentile); categorical data are presented as number (percentage).
A total of 5521 (4137 74.9% pacemaker and 1384 25.1% defibrillator) leads were extracted during 2999 TLE procedures (patient age 67.2 55.2, 76.2 years, 30.2% female). Lead implant duration was 4.7 (2.4, 8.3) years and 2.0 (1.0, 2.0) leads were extracted per procedure. Sixty-seven patients (2.2%) had died by 30 days after TLE. Variables with the highest predictive value for 30-day all-cause mortality included age, body mass index, hemoglobin, end-stage renal disease, left ventricular ejection fraction, New York Heart Association functional class, extraction for infection, number of prior lead extractions performed by the operator, and extraction of a dual-coil defibrillator lead. These variables were used to create a nomogram with a bootstrap-corrected concordance index value of 0.867.
Thirty-day all-cause mortality after TLE can be assessed with good discriminative power using readily available clinical information.
Light responses and photoadaptation of Neurospora depend on the photosensory light-oxygen-voltage (LOV) domains of the circadian transcription factor White Collar Complex (WCC) and its negative ...regulator VIVID (VVD). We found that light triggers LOV-mediated dimerization of the WCC. The activated WCC induces expression of VVD, which then disrupts and inactivates the WCC homodimers by the competitive formation of WCC-VVD heterodimers, leading to photoadaptation. During the day, expression levels of VVD correlate with light intensity, allowing photoadaptation over several orders of magnitude. At night, previously synthesized VVD serves as a molecular memory of the brightness of the preceding day and suppresses responses to light cues of lower intensity. We show that VVD is essential to discriminate between day and night, even in naturally ambiguous photoperiods with moonlight.
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► In Neurospora, light triggers dimerization of the circadian transcription factor WCC ► WCC induces expression of negative regulator VVD and formation of WCC-VVD dimers ► Light-correlated expression of VVD allows photoadaptation to changes in light levels ► VVD provides molecular memory of brightness, essential to discriminate day and night
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