Cardiac GR Mediates the Diurnal Rhythm in Ventricular Arrhythmia Susceptibility.

BACKGROUND: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.

PAK1-dependent mechanotransduction enables myofibroblast nuclear adaptation and chromatin organization during fibrosis.

Myofibroblasts are responsible for scarring during fibrosis. The scar propagates mechanical signals inducing a radical transformation in myofibroblast cell state and increasing profibrotic phenotype. Here, we show mechanical stress from progressive scarring induces nuclear softening and de-repression of heterochromatin. The parallel loss of H3K9Me3 enables a permissive state for distinct chromatin accessibility and profibrotic gene regulation. Integrating chromatin accessibility profiles with RNA expression provides insight into the transcription network underlying the switch in profibrotic myofibroblast states, emphasizing mechanoadaptive regulation of PAK1 as key drivers. Through genetic manipulation in liver and lung fibrosis, loss of PAK1-dependent signaling impairs the mechanoadaptive response in vitro and dramatically improves fibrosis in vivo. Moreover, we provide human validation for mechanisms underpinning PAK1-mediated mechanotransduction in liver and lung fibrosis. Collectively, these observations provide insight into the nuclear mechanics driving the profibrotic chromatin landscape in fibrosis, highlighting actomyosin-dependent mechanisms as potential therapeutic targets in fibrosis.

Circadian Disruption Primes Myofibroblasts for Accelerated Activation as a Mechanism Underpinning Fibrotic Progression in Non-Alcoholic Fatty Liver Disease.

Circadian rhythm governs many aspects of liver physiology and its disruption exacerbates chronic disease. CLOCKΔ19 mice disrupted circadian rhythm and spontaneously developed obesity and metabolic syndrome, a phenotype that parallels the progression of non-alcoholic fatty liver disease (NAFLD). NAFLD represents an increasing health burden with an estimated incidence of around 25% and is associated with an increased risk of progression towards inflammation, fibrosis and carcinomas. Excessive extracellular matrix deposition (fibrosis) is the key driver of chronic disease progression. However, little attention was paid to the impact of disrupted circadian rhythm in hepatic stellate cells (HSCs) which are the primary mediator of fibrotic ECM deposition. Here, we showed in vitro and in vivo that liver fibrosis is significantly increased when circadian rhythm is disrupted by CLOCK mutation. Quiescent HSCs from CLOCKΔ19 mice showed higher expression of RhoGDI pathway components and accelerated activation. Genes altered in this primed CLOCKΔ19 qHSC state may provide biomarkers for early liver disease detection, and include AOC3, which correlated with disease severity in patient serum samples. Integration of CLOCKΔ19 microarray data with ATAC-seq data from WT qHSCs suggested a potential CLOCK regulome promoting a quiescent state and downregulating genes involved in cell projection assembly. CLOCKΔ19 mice showed higher baseline COL1 deposition and significantly worse fibrotic injury after CCl4 treatment. Our data demonstrate that disruption to circadian rhythm primes HSCs towards an accelerated fibrotic response which worsens liver disease.

Aspergillus fumigatus Can Display Persistence to the Fungicidal Drug Voriconazole.

Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first-line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non-mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. IMPORTANCE The phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized the response of the prominent fungal pathogen Aspergillus fumigatus to the first-line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and propose various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.

Navigating the Global Protein-Protein Interaction Landscape Using iRefWeb.

iRefWeb is a resource that provides web interface to a large collection of protein-protein interactions aggregated from major primary databases. The underlying data-consolidation process, called iRefIndex, implements a rigorous methodology of identifying redundant protein sequences and integrating disparate data records that reference the same peptide sequences, despite many potential differences in data identifiers across various source databases. iRefWeb offers a unified user interface to all interaction records and associated information collected by iRefIndex, in addition to a number of data filters and visual features that present the supporting evidence. Users of iRefWeb can explore the consolidated landscape of protein-protein interactions, establish the provenance and reliability of each data record, and compare annotations performed by different data curator teams. The iRefWeb portal is freely available at http://wodaklab.org/iRefWeb .

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.

Dynamic changes in the epigenomic landscape regulate human organogenesis and link to developmental disorders.

How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development.

Vulnerability of drug-resistant EML4-ALK rearranged lung cancer to transcriptional inhibition.

A subset of lung adenocarcinomas is driven by the EML4-ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on-target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4-ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti-apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first-, second- and third-generation ALK inhibitors.

A living biobank of ovarian cancer ex vivo models reveals profound mitotic heterogeneity.

High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a "living biobank" of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making.

The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition.

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.

Combinatorial action of NF-Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish.

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF-Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF-Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF-Y or TALE defines genes in specific developmental processes, such that NF-Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF-Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF-Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.

Caring values in undergraduate nurse students: A qualitative longtitudinal study.

INTRODUCTION: Given the emerging evidence internationally of poor care within the healthcare sector, a recent report in the United Kingdom recommended the need for education to produce nurses who are prepared both intellectually and with compassion. AIM: This paper aims to understand the beliefs and values of caring, held by student nurses from entry to completion of their education programme. METHODS: Using a prospective qualitative longtitudinal approach, two cohorts of nursing students (February 2013 and 2014) each following a different undergraduate curriculum (the February 2013, based on a philosophy of person-centred care and the February 2014, based on the philosophy of humanisation) were followed throughout their programme leading to Registration. Data were collected from February 2013 to February 2017 using individual interviews at commencement and completion of their programme with focus groups after their first placement and at the end of years one and two. Using purposive sampling, from February 2013, 12 commenced the study and five finished. From February 2014, 24 started, with nine completing. FINDINGS: Data were analysed using thematic analysis with four themes emerging: i) Articulating the terms caring and dignity ii) Recognising the need for individualisation iii) Learning nursing and iv) Personal journey. CONCLUSION: Reporting on the final phase of this 5-phase study and on the brink of qualifying, both cohorts of students recognised the impact of their different curriculum and their exposure to the same educators who had embraced the humanisation philosophy. They each acknowledged just how they had changed as individuals and how determined they were to influence the quality of care.

The SmartTarget Biopsy Trial: A Prospective, Within-person Randomised, Blinded Trial Comparing the Accuracy of Visual-registration and Magnetic Resonance Imaging/Ultrasound Image-fusion Targeted Biopsies for Prostate Cancer Risk Stratification.

BACKGROUND: Multiparametric magnetic resonance imaging (mpMRI)-targeted prostate biopsies can improve detection of clinically significant prostate cancer and decrease the overdetection of insignificant cancers. It is unknown whether visual-registration targeting is sufficient or augmentation with image-fusion software is needed. OBJECTIVE: To assess concordance between the two methods. DESIGN, SETTING, AND PARTICIPANTS: We conducted a blinded, within-person randomised, paired validating clinical trial. From 2014 to 2016, 141 men who had undergone a prior (positive or negative) transrectal ultrasound biopsy and had a discrete lesion on mpMRI (score 3-5) requiring targeted transperineal biopsy were enrolled at a UK academic hospital; 129 underwent both biopsy strategies and completed the study. INTERVENTION: The order of performing biopsies using visual registration and a computer-assisted MRI/ultrasound image-fusion system (SmartTarget) on each patient was randomised. The equipment was reset between biopsy strategies to mitigate incorporation bias. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The proportion of clinically significant prostate cancer (primary outcome: Gleason pattern ≥3+4=7, maximum cancer core length ≥4mm; secondary outcome: Gleason pattern ≥4+3=7, maximum cancer core length ≥6mm) detected by each method was compared using McNemar's test of paired proportions. RESULTS AND LIMITATIONS: The two strategies combined detected 93 clinically significant prostate cancers (72% of the cohort). Each strategy detected 80/93 (86%) of these cancers; each strategy identified 13 cases missed by the other. Three patients experienced adverse events related to biopsy (urinary retention, urinary tract infection, nausea, and vomiting). No difference in urinary symptoms, erectile function, or quality of life between baseline and follow-up (median 10.5 wk) was observed. The key limitations were lack of parallel-group randomisation and a limit on the number of targeted cores. CONCLUSIONS: Visual-registration and image-fusion targeting strategies combined had the highest detection rate for clinically significant cancers. Targeted prostate biopsy should be performed using both strategies together. PATIENT SUMMARY: We compared two prostate cancer biopsy strategies: visual registration and image fusion. A combination of the two strategies found the most clinically important cancers and should be used together whenever targeted biopsy is being performed.

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.

Whole Genome Sequencing, de Novo Assembly and Phenotypic Profiling for the New Budding Yeast Species Saccharomyces jurei.

Saccharomyces sensu stricto complex consist of yeast species, which are not only important in the fermentation industry but are also model systems for genomic and ecological analysis. Here, we present the complete genome assemblies of Saccharomyces jurei, a newly discovered Saccharomyces sensu stricto species from high altitude oaks. Phylogenetic and phenotypic analysis revealed that S. jurei is more closely related to S. mikatae, than S. cerevisiae, and S. paradoxus The karyotype of S. jurei presents two reciprocal chromosomal translocations between chromosome VI/VII and I/XIII when compared to the S. cerevisiae genome. Interestingly, while the rearrangement I/XIII is unique to S. jurei, the other is in common with S. mikatae strain IFO1815, suggesting shared evolutionary history of this species after the split between S. cerevisiae and S. mikatae The number of Ty elements differed in the new species, with a higher number of Ty elements present in S. jurei than in S. cerevisiae Phenotypically, the S. jurei strain NCYC 3962 has relatively higher fitness than the other strain NCYC 3947T under most of the environmental stress conditions tested and showed remarkably increased fitness in higher concentration of acetic acid compared to the other sensu stricto species. Both strains were found to be better adapted to lower temperatures compared to S. cerevisiae.

TALE factors use two distinct functional modes to control an essential zebrafish gene expression program.

TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs.

Postmortem Genetic Testing for Cardiac Ion Channelopathies in Stillbirths.

BACKGROUND: Although stillbirth is a significant health problem worldwide, the definitive cause of death remains elusive in many cases, despite detailed autopsy. In this study of partly explained and unexplained stillbirths, we used next-generation sequencing to examine an extended panel of 35 candidate genes known to be associated with ion channel disorders and sudden cardiac death. METHODS AND RESULTS: We examined tissue from 242 stillbirths (≥22 weeks), including those where no definite cause of death could be confirmed after a full autopsy. We obtained high-quality DNA from 70 cases, which were then sequenced for a custom panel of 35 genes, 12 for inherited long- and short-QT syndrome genes (LQT1-LQT12 and SQT1-3), and 23 additional candidate genes derived from genome-wide association studies. We examined the functional significance of a selected variant by patch-clamp electrophysiological recording. No predicted damaging variants were identified in KCNQ1 (LQT1) or KCNH2 (LQT2). A rare putative pathogenic variant was found in KCNJ2(LQT7) in 1 case, and several novel variants of uncertain significance were observed. The KCNJ2 variant (p. R40Q), when assessed by whole-cell patch clamp, affected the function of the channel. There was no significant evidence of enrichment of rare predicted damaging variants within any of the candidate genes. CONCLUSIONS: Although a causative link is unclear, 1 putative pathogenic and variants of uncertain significance variant resulting in cardiac channelopathies was identified in some cases of otherwise unexplained stillbirth, and these variants may have a role in fetal demise. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01120886.