Safety and clinical impact of FEES - results of the FEES-registry.

BACKGROUND: At present, the flexible endoscopic evaluation of swallowing (FEES) is one of the most commonly used methods for the objective assessment of swallowing. This multicenter trial prospectively collected data on the safety of FEES and also assessed the impact of this procedure on clinical dysphagia management. METHODS: Patients were recruited in 23 hospitals in Germany and Switzerland from September 2014 to May 2017. Patient characteristics, professional affiliation of the FEES examiners (physicians or speech and language therapists), side-effects and cardiorespiratory parameters, severity of dysphagia and clinical consequences of FEES were documented. RESULTS: 2401 patients, mean age 69.8 (14.6) years, 42.3% women, were included in the FEES-registry. The most common main diagnosis was stroke (61%), followed by Parkinson's disease (6.5%). FEES was well tolerated by patients. Complications were reported in 2% of examinations, were all self-limited and resolved without sequelae and showed no correlation to the endoscopist's previous experience. In more than 50% of investigations FEES led to changes of feeding strategies, in the majority of cases an upgrade of oral diet was possible. DISCUSSION: This study confirmed that FEES, even when performed by less experienced clinicians is a safe and well tolerated procedure and significantly impacts on the patients' clinical course. Implementation of a FEES-service in different clinical settings may improve dysphagia care. TRIAL REGISTRATION: ClinicalTrials.gov NCT03037762, registered January 31st 2017.

Flexible endoscopic evaluation of swallowing (FEES) for neurogenic dysphagia: training curriculum of the German Society of Neurology and the German stroke society.

BACKGROUND: Neurogenic dysphagia is one of the most frequent and prognostically relevant neurological deficits in a variety of disorders, such as stroke, parkinsonism and advanced neuromuscular diseases. Flexible endoscopic evaluation of swallowing (FEES) is now probably the most frequently used tool for objective dysphagia assessment in Germany. It allows evaluation of the efficacy and safety of swallowing, determination of appropriate feeding strategies and assessment of the efficacy of different swallowing manoeuvres. The literature furthermore indicates that FEES is a safe and well-tolerated procedure. In spite of the huge demand for qualified dysphagia diagnostics in neurology, a systematic FEES education has not yet been established. RESULTS: The structured training curriculum presented in this article aims to close this gap and intends to enforce a robust and qualified FEES service. As management of neurogenic dysphagia is not confined to neurologists, this educational programme is applicable to other clinicians and speech-language therapists with expertise in dysphagia as well. CONCLUSION: The systematic education in carrying out FEES across a variety of different professions proposed by this curriculum will help to spread this instrumental approach and to improve dysphagia management.

The cellular bromodomain protein Brd4 has multiple functions in E2-mediated papillomavirus transcription activation.

The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription activation, is important for E2's transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2's interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+), a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV) life cycle.

Activation of SOX2 expression by BRD4-NUT oncogenic fusion drives neoplastic transformation in NUT midline carcinoma.

BRD4 is implicated in the pathogenesis of a number of different cancers. It is also the target of translocation t(15;19) that accounts for the highly aggressive NUT midline carcinoma (NMC). We discovered that t(15;19) NMC cells display the ability to grow into stem cell-like spheres and express an exceptionally high level of the stem cell marker, SOX2. The BRD4-NUT fusion oncogene resulting from t(15;19) translocation is required for the abnormal activation of SOX2, which drives the stem cell-like proliferation and cellular transformation in NMC cells. SOX2 knockdown phenocopies the effects of BRD4-NUT inhibition, whereas ectopic SOX2 expression rescues the phenotype. The BRD4-NUT-induced abnormal SOX2 activation was observed in multiple NMC cell lines as well as in NMC primary tumors. We further demonstrate that BRD4-NUT oncoprotein recruits p300 to stimulate transcription activation and that inhibition of p300 represses SOX2 transcription in NMC cells. These studies identify this stem cell marker as a novel BRD4-NUT target that supports the highly aggressive transforming activity of t(15;19) carcinomas. Our study provides new mechanistic insights for understanding how alteration of BRD4 function by BRD4-NUT oncogene leads to the highly malignant NMC carcinoma. Because abnormal stem cell self-renewal is frequently observed during tumor formation and metastasis, the aberrant stem cell-like proliferation associated with BRD4 dysregulation observed in NMC carcinoma may have implications for studying the oncogenic mechanism of other BRD4-associated tumors.

Analysis of the papillomavirus E2 and bromodomain protein Brd4 interaction using bimolecular fluorescence complementation.

The human papillomavirus (HPV) vaccines effectively protect against new infections of up to four HPV subtypes. However, these vaccines are not protective against many other clinically relevant HPV subtypes and are ineffective at treating established HPV infections. There is therefore a significant need for antiviral treatments for persistent HPV infections. A promising anti-HPV drug target is the interaction between the HPV E2 protein and cellular bromodomain-containing protein 4 (Brd4) since this protein complex mediates several processes important for the viral life cycle including viral genome maintenance, replication, and transcription. Using bimolecular fluorescence complementation (BiFC) technology, we demonstrate the E2 and Brd4 interaction on both interphase chromatin and mitotic chromosomes throughout mitosis. The E2-Brd4 BiFC was significantly diminished by mutating the Brd4 binding sites in E2 or by a dominant negative inhibitor of the E2-Brd4 interaction, demonstrating the potential of BiFC for identifying inhibitors of this important virus-host interaction. Importantly, when Brd4 was released from chromatin using the bromodomain inhibitor JQ1(+), the E2-Brd4 interacting complex relocated into foci that no longer associate with mitotic chromosomes, pointing to JQ1(+) as a promising antiviral inhibitor of HPV genome maintenance during HPV persistent infection.

Recruitment of Brd4 to the human papillomavirus type 16 DNA replication complex is essential for replication of viral DNA.

Replication of the human papillomavirus (HPV) DNA genome relies on viral factors E1 and E2 and the cellular replication machinery. Bromodomain-containing protein 4 (Brd4) interacts with viral E2 protein to mediate papillomavirus (PV) genome maintenance and viral transcription. However, the functional role of Brd4 in the HPV life cycle remains to be clearly defined. In this study, we provide the first look into the E2-Brd4 interaction in the presence of other important viral factors, such as the HPV16 E1 protein and the viral genome. We show that Brd4 is recruited to actively replicating HPV16 origin foci together with HPV16 E1, E2, and a number of the cellular replication factors: replication protein A70 (RPA70), replication factor C1 (RFC1), and DNA polymerase δ. Mutagenesis disrupting the E2-Brd4 interaction abolishes the formation of the HPV16 replication complex and impairs HPV16 DNA replication in cells. Brd4 was further demonstrated to be necessary for HPV16 viral DNA replication using a cell-free replication system in which depletion of Brd4 by small interfering RNA (siRNA) silencing leads to impaired HPV16 viral DNA replication and recombinant Brd4 protein is able to rescue viral DNA replication. In addition, releasing endogenous Brd4 from cellular chromatin by using the bromodomain inhibitor JQ1(+) enhances HPV16 DNA replication, demonstrating that the role of Brd4 in HPV DNA replication could be uncoupled from its function in chromatin-associated transcriptional regulation and cell cycle control. Our study reveals a new role for Brd4 in HPV genome replication, providing novel insights into understanding the life cycle of this oncogenic DNA virus.

Bromodomain protein Brd4 associated with acetylated chromatin is important for maintenance of higher-order chromatin structure.

Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture.