European Laryngological Society position paper on laryngeal dysplasia part II: diagnosis, treatment, and follow-up

Purpose of review To give an overview of the current knowledge regarding the diagnosis, treatment, and follow-up of laryngeal dysplasia (LD) and to highlight the contributions of recent literature. The diagnosis of LD largely relies on endoscopic procedures and on histopathology. Diagnostic efficiency of endoscopy may be improved using videolaryngostroboscopy (VLS) and bioendoscopic tools such as Narrow Band Imaging (NBI) or Storz Professional Image Enhancement System (SPIES). Current histological classifications are not powerful enough to clearly predict the risk to carcinoma evolution and technical issues such as sampling error, variation in epithelial thickness and inflammation hamper pathological examination. Almost all dysplasia grading systems are effective in different ways. The 2017 World Health Organization (WHO) system should prove to be an improvement as it is slightly more reproducible and easier for the non-specialist pathologist to apply. To optimize treatment decisions, surgeons should know how their pathologist grades samples and preferably audit their transformation rates locally. Whether carcinoma in situ should be used as part of such classification remains contentious and pathologists should agree with their clinicians whether they find this additional grade useful in treatment decisions. Recently, different studies have defined the possible utility of different biomarkers in risk classification. The main treatment modality for LD is represented by transoral laser microsurgery. Radiotherapy may be indicated in specific circumstances such as multiple recurrence or wide-field lesions. Medical treatment currently does not have a significant role in the management of LD. Follow-up for patients treated with LD is a fundamental part of their care and investigations may be supported by the same techniques used during diagnosis (VLS and NBI/SPIES).Otorhinolaryngolog

Cyclin-dependent kinase 18 controls trafficking of aquaporin-2 and its abundance through ubiquitin ligase STUB1, which functions as an AKAP

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure

An AKAP-Lbc-RhoA interaction inhibitor promotes the translocation of aquaporin-2 to the plasma membrane of renal collecting duct principal cells

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking

European white paper: oropharyngeal dysphagia in head and neck cancer

Purpose To develop a European White Paper document on oropharyngeal dysphagia (OD) in head and neck cancer (HNC). There are wide variations in the management of OD associated with HNC across Europe. Methods Experts in the management of specific aspects of OD in HNC across Europe were delegated by their professional medical and multidisciplinary societies to contribute to this document. Evidence is based on systematic reviews, consensus-based position statements, and expert opinion. Results Twenty-four sections on HNC-specific OD topics. Conclusion This European White Paper summarizes current best practice on management of OD in HNC, providing recommendations to support patients and health professionals. The body of literature and its level of evidence on diagnostics and treatment for OD in HNC remain poor. This is in the context of an expected increase in the prevalence of OD due to HNC in the near future. Contributing factors to increased prevalence include aging of our European population (including HNC patients) and an increase in human papillomavirus (HPV) related cancer, despite the introduction of HPV vaccination in various countries. We recommend timely implementation of OD screening in HNC patients while emphasizing the need for robust scientific research on the treatment of OD in HNC. Meanwhile, its management remains a challenge for European professional associations and policymakers.Otorhinolaryngolog

Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains

PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg(173), Asn(174) and Asn(175) (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was additionally required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp(463) and Arg(499) in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, because residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure

Disruptors of AKAP-dependent protein-protein interactions

A-kinase anchoring proteins (AKAPs) are a family of multivalent scaffolding proteins. They engage in direct protein-protein interactions with protein kinases, kinase substrates and further signaling molecules. Each AKAP interacts with a specific set of protein interaction partners and such sets can vary between different cellular compartments and cells. Thus, AKAPs can coordinate signal transduction processes spatially and temporally in defined cellular environments. AKAP-dependent protein-protein interactions are involved in a plethora of physiological processes, including processes in the cardiovascular, nervous, and immune system. Dysregulation of AKAPs and their interactions is associated with or causes widespread diseases, for example, cardiac diseases such as heart failure. However, there are profound shortcomings in understanding functions of specific AKAP-dependent protein-protein interactions. In part, this is due to the lack of agents for specifically targeting defined protein-protein interactions. Peptidic and non-peptidic inhibitors are invaluable molecular tools for elucidating the functions of AKAP-dependent protein-protein interactions. In addition, such interaction disruptors may pave the way to new concepts for the treatment of diseases where AKAP-dependent protein-protein interactions constitute potential drug targets.Here we describe screening approaches for the identification of small molecule disruptors of AKAP-dependent protein-protein interactions. Examples include interactions of AKAP18 and protein kinase A (PKA) and of AKAP-Lbc and RhoA. We discuss a homogenous time-resolved fluorescence (HTRF) and an AlphaScreen® assay for small molecule library screening and human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) as a cell system for the characterization of identified hits

Gravity and Mastoid Effusion

Background: Asymptomatic mastoid effusions have recently been observed in astronauts returning from long-term spaceflight. In hospitalized patients, mastoid effusion increases the risks for bacterial otitis and mastoiditis. We reasoned that cephalad fluid shifts during strict -6° head down tilt bed rest could reproduce space-flight associated mastoid effusion and that artificial gravity may reverse the response. Methods: The recent Artificial Gravity Bed Rest Study-European Space Agency study (AGBRESA) tested influences of artificial gravity during 60 days head down bed rest on a short-arm human centrifuge in healthy participants. The two intervention groups received daily artificial gravity with 30 minutes continuous artificial gravity or intermittent artificial gravity. A third group served as a control group and received no artificial gravity. We assessed cranial magnetic resonance images for mastoid effusions 1 day before bed rest, at days 14 and 52 of bed rest, and 3 days after bed rest. Results: None of the participants exhibited mastoid effusions before bed rest. Six participants showed mastoid effusions at bed rest day 14 (4 continuous, 2 intermittent, 0 control). Fifteen participants showed mastoid effusions at bed rest day 52 and 3 days after bed rest (7 continuous, 3 intermittent, 5 control). Conclusions: Mastoid effusions commonly occur during strict head down tilt bed rest. The model can be applied to study the mechanisms and potential countermeasures for space flight-associated mastoid effusions. Formation of mastoid effusions during head down tilt bed rest is not prevented by daily 30 minutes short-arm intermittent or continuous centrifugation

Aurora kinase A is involved in controlling the localization of aquaporin-2 in renal principal cells

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown