Chronic inflammatory arthritis drives systemic changes in circadian energy metabolism.

Chronic inflammation underpins many human diseases. Morbidity and mortality associated with chronic inflammation are often mediated through metabolic dysfunction. Inflammatory and metabolic processes vary through circadian time, suggesting an important temporal crosstalk between these systems. Using an established mouse model of rheumatoid arthritis, we show that chronic inflammatory arthritis results in rhythmic joint inflammation and drives major changes in muscle and liver energy metabolism and rhythmic gene expression. Transcriptional and phosphoproteomic analyses revealed alterations in lipid metabolism and mitochondrial function associated with increased EGFR-JAK-STAT3 signaling. Metabolomic analyses confirmed rhythmic metabolic rewiring with impaired ß-oxidation and lipid handling and revealed a pronounced shunt toward sphingolipid and ceramide accumulation. The arthritis-related production of ceramides was most pronounced during the day, which is the time of peak inflammation and increased reliance on fatty acid oxidation. Thus, our data demonstrate that localized joint inflammation drives a time-of-day­dependent build-up of bioactive lipid species driven by rhythmic inflammation and altered EGFR-STAT signaling.

Circadian clock function does not require the histone methyltransferase MLL3.

The circadian clock controls the physiological function of tissues through the regulation of thousands of genes in a cell-type-specific manner. The core cellular circadian clock is a transcription-translation negative feedback loop, which can recruit epigenetic regulators to facilitate temporal control of gene expression. Histone methyltransferase, mixed lineage leukemia gene 3 (MLL3) was reported to be required for the maintenance of circadian oscillations in cultured cells. Here, we test the role of MLL3 in circadian organization in whole animals. Using mice expressing catalytically inactive MLL3, we show that MLL3 methyltransferase activity is in fact not required for circadian oscillations in vitro in a range of tissues, nor for the maintenance of circadian behavioral rhythms in vivo. In contrast to a previous report, loss of MLL3-dependent methylation did not affect the global levels of H3K4 methylation in liver, indicating substantial compensation from other methyltransferases. Furthermore, we found little evidence of genomic repositioning of H3K4me3 marks. We did, however, observe repositioning of H3K4me1 from intronic regions to intergenic regions and gene promoters; however, there were no changes in H3K4me1 mark abundance around core circadian clock genes. Output functions of the circadian clock, such as control of inflammation, were largely intact in MLL3-methyltransferase-deficient mice, although some gene-specific changes were observed, with sexually dimorphic loss of circadian regulation of specific cytokines. Taken together, these observations indicate that MLL3-directed histone methylation is not essential for core circadian clock function; however, it may influence the inflammatory response.

The clock gene Bmal1 inhibits macrophage motility, phagocytosis, and impairs defense against pneumonia.

The circadian clock regulates many aspects of immunity. Bacterial infections are affected by time of day, but the mechanisms involved remain undefined. Here we show that loss of the core clock protein BMAL1 in macrophages confers protection against pneumococcal pneumonia. Infected mice show both reduced weight loss and lower bacterial burden in circulating blood. In vivo studies of macrophage phagocytosis reveal increased bacterial ingestion following Bmal1 deletion, which was also seen in vitro. BMAL1-/- macrophages exhibited marked differences in actin cytoskeletal organization, a phosphoproteome enriched for cytoskeletal changes, with reduced phosphocofilin and increased active RhoA. Further analysis of the BMAL1-/- macrophages identified altered cell morphology and increased motility. Mechanistically, BMAL1 regulated a network of cell movement genes, 148 of which were within 100 kb of high-confidence BMAL1 binding sites. Links to RhoA function were identified, with 29 genes impacting RhoA expression or activation. RhoA inhibition restored the phagocytic phenotype to that seen in control macrophages. In summary, we identify a surprising gain of antibacterial function due to loss of BMAL1 in macrophages, associated with a RhoA-dependent cytoskeletal change, an increase in cell motility, and gain of phagocytic function.

Circadian rhythms in innate immunity and stress responses.

Circadian clocks are a common feature of life on our planet, allowing physiology and behaviour to be adapted to recurrent environmental fluctuation. There is now compelling evidence that disturbance of circadian coherence can severely undermine mental and physical health, as well as exacerbate pre-existing pathology. Common molecular design principles underpin the generation of cellular circadian rhythms across the kingdoms, and in animals, the genetic components are extremely well conserved. In mammals, the circadian timing mechanism is present in most cell types and establishes local cycles of gene expression and metabolic activity. These distributed tissue clocks are normally synchronized by a central pacemaker, the suprachiasmatic nuclei (SCN), located in the hypothalamus. Nevertheless, most clocks of the body remain responsive to non-SCN-derived hormonal and metabolic cues (for example, re-alignment of liver clocks to altered meal patterning). It has been demonstrated that the clock is an influential regulator of energy metabolism, allowing key pathways to be tuned across the 24-hr cycle as metabolic requirements fluctuate. Furthermore, clock components, including Cryptochrome and Rev-Erb proteins, have been identified as essential modulators of the innate immune system and inflammatory responses. Studies have also revealed that these proteins regulate glucocorticoid receptor function, a major drug target and crucial regulator of inflammation and metabolism.

Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Pulmonary airway epithelial cells (AECs) form a critical interface between host and environment. We investigated the role of the circadian clock using mice bearing targeted deletion of the circadian gene brain and muscle ARNT-like 1 (Bmal1) in AECs. Pulmonary neutrophil infiltration, biomechanical function, and responses to influenza infection were all disrupted. A circadian time-series RNA sequencing study of laser-captured AECs revealed widespread disruption in genes of the core circadian clock and output pathways regulating cell metabolism (lipids and xenobiotics), extracellular matrix, and chemokine signaling, but strikingly also the gain of a novel rhythmic transcriptome in Bmal1-targeted cells. Many of the rhythmic components were replicated in primary AECs cultured in air-liquid interface, indicating significant cell autonomy for control of pulmonary circadian physiology. Finally, we found that metabolic cues dictate phasing of the pulmonary clock and circadian responses to immunologic challenges. Thus, the local circadian clock in AECs is vital in lung health by coordinating major cell processes such as metabolism and immunity.-Zhang, Z. Hunter, L., Wu, G., Maidstone, R., Mizoro, Y., Vonslow, R., Fife, M., Hopwood, T., Begley, N., Saer, B., Wang, P., Cunningham, P., Baxter, M., Durrington, H., Blaikley, J. F., Hussell, T., Rattray, M., Hogenesch, J. B., Gibbs, J., Ray, D. W., Loudon, A. S. I. Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Glucocorticoid receptor function in health and disease.

Glucocorticoid hormones are essential for life in vertebrates. They act through the glucocorticoid receptor (GR), which is expressed in virtually all cells of the human body. Yet the actions of glucocorticoids (GCs) are specific to particular cell types. Broadly GCs regulate carbohydrate metabolism, inflammation, stress and cell fate. Synthetic GCs are widely used in medicine and are by far the most frequent cause of Cushing's syndrome in routine practice. The advent of novel drugs targeting the GR offers new opportunities to treat patients with immune, or malignant disease, and may also offer new opportunities to manage patients with adrenal insufficiency also. This review covers the latest understanding of how GCs work, how their actions are affected by disease, and where the new drugs may take us.

A General Access to Propargylic Ethers through Brønsted Acid Catalyzed Alkynylation of Acetals and Ketals with Trifluoroborates†.

A general Brønsted acid catalyzed methodology for the alkynylation of acetals and ketals with alkynyltrifluoroborate salts has been developed. The reaction proceeds rapidly to afford valuable synthetic building block propargylic ethers in good to excellent yields. Unlike Lewis acid catalyzed transformations of trifluoroborates, this approach does not proceed via unstable organodifluoroborane intermediate. As a result, the developed methodology features excellent functional group tolerance and good atom economy.

Role of transient receptor potential and pannexin channels in cigarette smoke-triggered ATP release in the lung.

BACKGROUND: COPD is an inflammatory disease usually associated with cigarette smoking (CS) with an increasing global prevalence and no effective medication. Extracellular ATP is increased in the COPD affected lung and may play a key role in driving CS-induced airway inflammation, but the mechanism involved in ATP release has eluded researchers. Recently, the transient receptor potential (TRP) and pannexin-1 channels have been suggested to play a role in other experimental paradigms. Thus, the aim of this work is to investigate if these channels are involved in CS-induced ATP release in the lung. METHODS: Primary human cells were exposed to CS and extracellular ATP levels measured. Mice were exposed to mainstream CS and airway inflammation assessed. TRPV1/4 mRNA expression was assessed in human lung parenchyma. RESULTS: CS exposure caused a dose-related increase in ATP from primary airway bronchial epithelial cells. This was attenuated by blockers of TRPV1, TRPV4 and pannexin-1 channels. Parallel data was obtained using murine acute CS-driven model systems. Finally, TRPV1/4 mRNA expression was increased in lung tissue samples from patients with COPD. CONCLUSIONS: Extracellular ATP is increased in the COPD affected lung and may play a key role in driving disease pathophysiology. These experiments uncover a novel mechanism which may be responsible for CS-induced ATP release. These findings highlight novel targets that could lead to the development of medicine to treat this devastating disease.

Shift work and evening chronotype are associated with hepatic fat fraction and non-alcoholic fatty liver disease in 282,303 UK biobank participants.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) has rapidly become the most common liver disease worldwide. Modern lifestyles have been linked to this rise in prevalence with changes in rhythmic human behaviour emerging as a possible mechanism. We investigated how shift working patterns and chronotype were associated with hepatic fat fraction and NAFLD in 282,303 UK Biobank participants. Methods: We stratified participants into day, irregular-shift, and permanent night-shift workers. We then utilised multiple methods of disease identification including (i) Dallas steatosis index (DSI), (ii) ICD10 codes, and (iii) hepatic proton density fat fraction (PDFF) and examined how shift work exposure impacted these variables. We further assessed the relationship of baseline chronotype with liver phenotypes using these same outcome measures. Results: Compared to day workers, irregular-shift workers were more likely to have a high DSI (OR 1.29 (1.2-1.4)) after adjusting for major covariates with some attenuation after additional adjustment for BMI (OR 1.12 (1.03-1.22)). Likelihood of high DSI was also increased in permanent night-shift workers (OR 1.08 (0.9-1.29)) in the fully adjusted model. Mediator analysis revealed that BMI was a significant mediator of the shift work effect. Compared to participants with intermediate chronotype, those with extreme late chronotype had a higher likelihood of high DSI defined NAFLD (OR 1.45 (1.34-1.56)) and a higher likelihood of NAFLD/NASH by ICD10 code (OR 1.23 (1.09-1.39)). Hepatic PDFF was elevated in irregular shift workers, but not permanent night-shift workers. Conclusions: Irregular-shift work and extreme late chronotype are associated with pathological liver fat accumulation, suggesting circadian misalignment may have an underlying pathogenic role. These findings have implications for health interventions to mitigate the detrimental effect of shift work.

The interplay between macronutrients and sleep: focus on circadian and homeostatic processes.

Sleep disturbances are an emerging risk factor for metabolic diseases, for which the burden is particularly worrying worldwide. The importance of sleep for metabolic health is being increasingly recognized, and not only the amount of sleep plays an important role, but also its quality. In this review, we studied the evidence in the literature on macronutrients and their influence on sleep, focusing on the mechanisms that may lay behind this interaction. In particular, we focused on the effects of macronutrients on circadian and homeostatic processes of sleep in preclinical models, and reviewed the evidence of clinical studies in humans. Given the importance of sleep for health, and the role of circadian biology in healthy sleep, it is important to understand how macronutrients regulate circadian clocks and sleep homeostasis.

CATCH-UP: A High-Throughput Upstream-Pipeline for Bulk ATAC-Seq and ChIP-Seq Data.

Assay for transposase-accessible chromatin (ATAC) and chromatin immunoprecipitation (ChIP), coupled with next-generation sequencing (NGS), have revolutionized the study of gene regulation. A lack of standardization in the analysis of the highly dimensional datasets generated by these techniques has made reproducibility difficult to achieve, leading to discrepancies in the published, processed data. Part of this problem is due to the diverse range of bioinformatic tools available for the analysis of these types of data. Secondly, a number of different bioinformatic tools are required sequentially to convert raw data into a fully processed and interpretable output, and these tools require varying levels of computational skills. Furthermore, there are many options for quality control that are not uniformly employed during data processing. We address these issues with a complete assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) upstream pipeline (CATCH-UP), an easy-to-use, Python-based pipeline for the analysis of bulk ChIP-seq and ATAC-seq datasets from raw fastq files to visualizable bigwig tracks and peaks calls. This pipeline is simple to install and run, requiring minimal computational knowledge. The pipeline is modular, scalable, and parallelizable on various computing infrastructures, allowing for easy reporting of methodology to enable reproducible analysis of novel or published datasets.

Amphipathic antimicrobial piscidin in magnetically aligned lipid bilayers.

The amphipathic antimicrobial peptide piscidin 1 was studied in magnetically aligned phospholipid bilayers by oriented-sample solid-state NMR spectroscopy. (31)P NMR and double-resonance (1)H/(15)N NMR experiments performed between 25 °C and 61 °C enabled the lipid headgroups as well as the peptide amide sites to be monitored over a range of temperatures. The α-helical peptide dramatically affects the phase behavior and structure of anionic bilayers but not those of zwitterionic bilayers. Piscidin 1 stabilizes anionic bilayers, which remain well aligned up to 61 °C when piscidin 1 is on the membrane surface. Two-dimensional separated-local-field experiments show that the tilt angle of the peptide is 80 ± 5°, in agreement with previous results on mechanically aligned bilayers. The peptide undergoes fast rotational diffusion about the bilayer normal under these conditions, and these studies demonstrate that magnetically aligned bilayers are well suited for structural studies of amphipathic peptides.

A simple equation to correct for gadolinium interference on plasma selenium measurement using inductively coupled plasma mass spectrometry.

BACKGROUND: The measurement of selenium in human plasma is useful to assess deficiency or toxicity. The presence of gadolinium in clinical samples following administration of certain contrast agents used for magnetic resonance imaging can cause a significant positive bias in selenium results when measured using quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS). METHODS: A mathematical equation to correct for gadolinium interference was assessed using both patient samples and commercial quality control/external quality assurance (QC/EQA) materials spiked with gadolinium. Samples were analysed using an Agilent 7900 ICP-MS operated in 'narrow peak' (half-mass) mode. Accuracy was evaluated by comparing corrected selenium results with target concentrations. RESULTS: Corrected results were found to be accurate at all gadolinium concentrations tested (2, 4, 10 and 20 mg/L). Average recoveries ranged from 97.4 to 106.5%. Results for QC/EQA materials were within specified target ranges. Within-run imprecision was <3%, and between-run imprecision was <4.3%, demonstrating robustness. CONCLUSIONS: The correction equation described here is a simple method to correct for gadolinium interference on plasma selenium measurement using ICP-MS. This approach eliminates the need for specimen recollections, and improves patient care by reducing laboratory turnaround times and preventing delays in diagnosis/treatment.

Hypoxia regulates GR function through multiple mechanisms involving microRNAs 103 and 107.

Glucocorticoids (Gcs) potently inhibit inflammation, and regulate liver energy metabolism, often acting in a hypoxic environment. We now show hypoxic conditions open a specific GR cistrome, and prevent access of GR to part of the normoxic GR cistrome. Motif analysis identified enrichment of KLF4 binding sites beneath those peaks of GR binding exclusive to normoxia, implicating KLF4 as a pioneer, or co-factor under these conditions. Hypoxia reduced KLF4 expression, however, knockdown of KLF4 did not impair GR recruitment. KLF4 is a known target of microRNAs 103 and 107, both of which are induced by hypoxia. Expression of mimics to either microRNA103, or microRNA107 inhibited GR transactivation of normoxic target genes, thereby replicating the hypoxic effect. Therefore, studies in hypoxia reveal that microRNAs 103 and 107 are potent regulators of GR function. We have now identified a new pathway linking hypoxia through microRNAs 103 and 107 to regulation of GR function.

Cardiac mitochondrial function depends on BUD23 mediated ribosome programming.

Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells. Deletion of Bud23 in murine cardiomyocytes reduced mitochondrial content and function, leading to severe cardiomyopathy and death. We discovered that BUD23 selectively promotes ribosomal interaction with low GC-content 5'UTRs. Taken together we identify a critical role for BUD23 in bioenergetics gene expression, by promoting efficient translation of mRNA transcripts with low 5'UTR GC content. BUD23 emerges as essential to mouse development, and to postnatal cardiac function.

Cells need to make proteins to survive, so they have protein-making machines called ribosomes. Ribosomes are themselves made out of proteins and RNA (a molecule similar to DNA), and they are assembled by other proteins that bring ribosomal components together and modify them until the ribosomes are functional.Mitochondria are compartments in the cell that are in charge of providing it with energy. To do this they require several proteins produced by the ribosomes. If not enough mitochondrial proteins are made, mitochondria cannot provide enough energy for the cell to survive.One of the proteins involved in modifying ribosomes so they are functional is called BUD23. People with certain diseases, such as Williams-Beuren syndrome, do not make enough BUD23; but it was unknown what specific effects resulted from a loss of BUD23.To answer this question, Baxter et al. first genetically removed BUD23 from human cells, and then checked what happened to protein production. They found that ribosomes in human cells with no BUD23 were different than in normal cells, and that cells without BUD23 produced different proteins, which did not always perform their roles correctly. Proteins in the mitochondria are one of the main groups affected by the absence of BUD23. To determine what effects these modified mitochondrial proteins would have in an animal, Baxter et al. genetically modified mice so that they no longer produced BUD23. These mice developed heart problems caused by their mitochondria not working correctly and being unable to provide the energy the heart cells needed, eventually leading to heart failure. Heart problems are common in people with Williams-Beuren syndrome.Many diseases arise when a person's mitochondria do not work properly, but it is often unclear why. These experiments suggest that low levels of BUD23 or faulty ribosomes may be causing mitochondria to work poorly in some of these diseases, which could lead to the development of new therapies.

Inductively Coupled Plasma Mass Spectrometry: Introduction to Analytical Aspects.

Inductively coupled plasma mass spectrometry (ICP-MS) is an analytical technique that can be used to measure elements at trace levels in biological fluids. Although older techniques such as atomic absorption and atomic emission are still in use by some laboratories, there has been a slow shift toward ICP-MS, particularly in the last decade. As this shift is likely to continue, clinical scientists should be aware of the analytical aspects of ICP-MS, as well as the potential for both spectroscopic and non-spectroscopic interference, and strategies that can be employed to eliminate or mitigate these issues.

An improved method for quantitative ChIP studies of nuclear receptor function.

Chromatin immunoprecipitation (ChIP) is a valuable tool for the endocrine researcher, providing a means to measure the recruitment of hormone-activated nuclear receptors, for example. However, the technique can be challenging to perform and has multiple experimental steps, risking introduction of error at each. The data produced can be challenging to interpret; several different methods are commonly used for normalising data and thus comparing between conditions. Absolute, sensitive quantification of protein-bound DNA is important for correct interpretation of the data. In addition, such quantification can help the investigator in troubleshooting experiments. Here, we outline a ChIP strategy combining droplet digital PCR for accurate quantification with an internal spike-in control for normalisation. This combination strengthens the reliability of ChIP data and allows the operator to optimise their protocol with greater confidence.

REVERBa couples the circadian clock to hepatic glucocorticoid action.

The glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis - all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism.