Systematic review of the diagnosis and management of necrotising otitis externa: Highlighting the need for high-quality research.

OBJECTIVES: To present a systematic review and critical analysis of clinical studies for necrotising otitis externa (NOE), with the aim of informing best practice for diagnosis and management. DESIGN: Medline, Embase, Cochrane Library and Web of Science were searched from database inception until 30 April 2021 for all clinical articles on NOE. The review was registered on PROSPERO (ID: CRD42020128957) and conducted in accordance with PRISMA guidelines. RESULTS: Seventy articles, including 2274 patients were included in the final synthesis. Seventy-three percent were retrospective case series; the remainder were of low methodological quality. Case definitions varied widely. Median patient age was 69.2 years; 68% were male, 84% had diabetes and 10% had no reported immunosuppressive risk factor. Otalgia was almost universal (96%), with granulation (69%) and oedema (76%) the commonest signs reported. Pseudomonas aeruginosa was isolated in 62%, but a range of bacterial and fungal pathogens were reported and 14% grew no organism. Optimal imaging modality for diagnosis or follow-up was unclear. Median antimicrobial therapy duration was 7.2 weeks, with no definitive evidence for optimal regimens. Twenty-one percent had surgery with widely variable timing, indication, or procedure. One-year disease-specific mortality was 2%; treatment failure and relapse rates were 22% and 7%, respectively. CONCLUSION: There is a lack of robust, high-quality data to support best practice for diagnosis and management for this neglected condition. A minimum set of reporting requirements is proposed for future studies. A consensus case definition is urgently needed to facilitate high-quality research.

A retrospective cohort study of telephone versus face-to-face clinics for the management of new otology referrals.

PURPOSE: To compare outcomes of telephone and face-to-face consultations for new otology referrals and discuss the wider use of telemedicine in otology. METHODS: Retrospective cohort study including new adult otology referrals to our unit, sampled consecutively between March 2021 and May 2021, seen in either a face-to-face or telephone clinic. Primary outcome measure was the proportion of patients with a definitive management outcome (discharged or added to waiting list for treatment) versus the proportion of patients requiring follow-up for further assessment or review. RESULTS: 150 new patients referred for a routine otology consultation (75 telephone, 75 face-to-face) were included. 53/75 patients (71%) undergoing a face-to-face consultation received a definitive outcome following initial review, versus 22/75 (29%) telephone patients (χ2 < 0.001, OR 5.8). 52/75 (69%) telephone patients were followed up face-to-face for examination. The mean (SD) number of appointments required to reach a definitive outcome was 1.22 (0.58) and 1.75 (0.73) in the face-to-face and telephone cohorts, respectively (p < 0.001). CONCLUSIONS: Telephone clinics in otology have played an important role as part of the COVID19 response. However, they are currently limited by a lack of clinical examination and audiometry. Remote assessment pathways in otology that incorporate asynchronous review of recorded examinations alongside audiometry, either conventional or boothless, may mitigate this problem; however, further research is required.

Inactivation of Ppp1r15a minimises weight gain and insulin resistance during caloric excess in female mice.

Phosphorylation of the translation initiation factor eIF2α within the mediobasal hypothalamus is known to suppress food intake, but the role of the eIF2α phosphatases in regulating body weight is poorly understood. Mice deficient in active PPP1R15A, a stress-inducible eIF2α phosphatase, are healthy and more resistant to endoplasmic reticulum stress than wild type controls. We report that when female Ppp1r15a mutant mice are fed a high fat diet they gain less weight than wild type littermates owing to reduced food intake. This results in healthy leaner Ppp1r15a mutant animals with reduced hepatic steatosis and improved insulin sensitivity, albeit with a possible modest defect in insulin secretion. By contrast, no weight differences are observed between wild type and Ppp1r15a deficient mice fed a standard diet. We conclude that female mice lacking the C-terminal PP1-binding domain of PPP1R15A show reduced dietary intake and preserved glucose tolerance. Our data indicate that this results in reduced weight gain and protection from diet-induced obesity.

Assessment of Smell Function in Syndromic Craniosynostosis Patients.

Craniosynostosis is defined as premature fusion of the cranial suture lines and is part of a syndrome in 15% to 40% of the patients. There is limited literature available regarding these children's ability to smell. Most of them will undergo numerous surgical procedures, some of which may alter their sense of smell, potentially leading to significant social as well as safety implications. Ethical approval was obtained for this pilot study. Children with syndromic craniosynostosis were recruited and underwent anterior rhinoscopy, prior to performing a smell test utilizing the Sensonic pediatric Smell wheel. The results were compared to an age-matched control group. Eight children with syndromic craniosynostosis participated in the study. Of a possible total score of 11, their mean average score was 6.6 and the median was 6. In comparison, the mean average score for the control group was 7.5 and the median was 7. Although the study group was small, this pilot study demonstrates that children with syndromic craniosynostosis have a similar ability to identify smells to an age-matched cohort. Further research can now be undertaken to see whether or not midface advancement procedures affect these children's sense of smell.

Interactions between N-linked glycosylation and polymerisation of neuroserpin within the endoplasmic reticulum.

The neuronal serpin neuroserpin undergoes polymerisation as a consequence of point mutations that alter its conformational stability, leading to a neurodegenerative dementia called familial encephalopathy with neuroserpin inclusion bodies (FENIB). Neuroserpin is a glycoprotein with predicted glycosylation sites at asparagines 157, 321 and 401. We used site-directed mutagenesis, transient transfection, western blot, metabolic labelling and ELISA to probe the relationship between glycosylation, folding, polymerisation and degradation of neuroserpin in validated cell models of health and disease. Our data show that glycosylation at N157 and N321 plays an important role in maintaining the monomeric state of neuroserpin, and we propose this is the result of steric hindrance or effects on local conformational dynamics that can contribute to polymerisation. Asparagine residue 401 is not glycosylated in wild type neuroserpin and in several polymerogenic variants that cause FENIB, but partial glycosylation was observed in the G392E mutant of neuroserpin that causes severe, early-onset dementia. Our findings indicate that N401 glycosylation reports lability of the C-terminal end of neuroserpin in its native state. This C-terminal lability is not required for neuroserpin polymerisation in the endoplasmic reticulum, but the additional glycan facilitates degradation of the mutant protein during proteasomal impairment. In summary, our results indicate how normal and variant-specific N-linked glycosylation events relate to intracellular folding, misfolding, degradation and polymerisation of neuroserpin.

Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.

Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation.

Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR.

Coordinate regulation of eIF2α phosphorylation by PPP1R15 and GCN2 is required during Drosophila development.

Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by the kinase GCN2 attenuates protein synthesis during amino acid starvation in yeast, whereas in mammals a family of related eIF2α kinases regulate translation in response to a variety of stresses. Unlike single-celled eukaryotes, mammals also possess two specific eIF2α phosphatases, PPP1R15a and PPP1R15b, whose combined deletion leads to a poorly understood early embryonic lethality. We report the characterisation of the first non-mammalian eIF2α phosphatase and the use of Drosophila to dissect its role during development. The Drosophila protein demonstrates features of both mammalian proteins, including limited sequence homology and association with the endoplasmic reticulum. Of note, although this protein is not transcriptionally regulated, its expression is controlled by the presence of upstream open reading frames in its 5'UTR, enabling induction in response to eIF2α phosphorylation. Moreover, we show that its expression is necessary for embryonic and larval development and that this is to oppose the inhibitory effects of GCN2 on anabolic growth.

Constitutive RAC activation is not sufficient to initiate melanocyte neoplasia but accelerates malignant progression.

Deregulated Ras signaling initiates and maintains melanocyte neoplasia. The Rho-like GTPase Rac has been implicated in Ras-induced neoplastic transformation. Moreover, a recurrent UV-induced mutation activating RAC1 has recently been detected in human melanoma. Here, a role for Rac in melanoma initiation and progression was investigated in human melanomas and zebrafish models of melanocyte neoplasia. Immunohistochemical analysis revealed RAC expression and activity restricted to melanocytes at the junction of the epidermis and dermis in benign neoplasms. Malignant melanocytes displayed elevated RAC activity that extended into the suprabasal epidermis, deeper into the dermis, and was maintained in metastases. Previously, we have used zebrafish transgenic models to demonstrate that deregulated Ras/Raf/mitogen-activated protein kinase signaling can initiate melanocyte neoplasia. Expression of a constitutively active RAC1 mutant (V12RAC1) was not sufficient to initiate melanocyte neoplasia in this organism. Furthermore, we did not detect an additive effect when combined with V600EBRAF, nor could V12RAC1 substitute for suppressed Pi3k signaling to restore melanoma progression. However, coexpression of V12RAC1 and oncogenic RAS accelerated tumor nodule formation. Immunohistochemical analysis revealed that the Rac activator Tiam1 (T-cell lymphoma invasion and metastasis 1) is overexpressed in melanoma tumor nodules in both zebrafish and humans. Thus, our data suggest that Rac contributes to the progression of melanoma and that Tiam1 may activate Rac in nodular presentations.

p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress.

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G(1) phase, although recent studies have identified a novel ER stress G(2) checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G(2) delay involving CHK1 and a later induction of G(1) arrest associated both with the induction of p53 target genes and loss of cyclin D(1). We show that substitution of p53/47 for p53 impairs the ER stress G(1) checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G(2) progression prior to ultimate G(1) arrest.

Phosphoproteins in stress-induced disease.

The integrated stress response (ISR) is an evolutionarily conserved homeostatic program activated by specific pathological states. These include amino acid deprivation, viral infection, iron deficiency, and the misfolding of proteins within the endoplasmic reticulum (ER), the so-called ER stress. Although apparently disparate, each of these stresses induces phosphorylation of a translation initiation factor, eIF2α, to attenuate new protein translation while simultaneously triggering a transcriptional program. This is achieved by four homologous stress-sensing kinases: GCN2, PKR, HRI, and PERK. In addition to these kinases, mammals possess two specific eIF2α phosphatases, GADD34 and CReP, which play crucial roles in the recovery of protein synthesis following the initial insult. They are not only important in embryonic development but also appear to play important roles in disease, particularly cancer. In this chapter, we discuss each of the eIF2α kinases, in turn, with particular emphasis on their regulation and the new insights provided by recent structural studies. We also discuss the potential for developing novel drug therapies that target the ISR.

The serpinopathies studying serpin polymerization in vivo.

The serpinopathies result from point mutations in members of the serine protease inhibitor or serpin superfamily. They are characterized by the formation of ordered polymers that are retained within the cell of synthesis. This causes disease by a "toxic gain of function" from the accumulated protein and a "loss of function" as a result of the deficiency of inhibitors that control important proteolytic cascades. The serpinopathies are exemplified by the Z (Glu342Lys) mutant of α1-antitrypsin that results in the retention of ordered polymers within the endoplasmic reticulum of hepatocytes. These polymers form the intracellular inclusions that are associated with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. A second example results from mutations in the neurone-specific serpin-neuroserpin to form ordered polymers that are retained as inclusions within subcortical neurones as Collins' bodies. These inclusions underlie the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. There are different pathways to polymer formation in vitro but not all form polymers that are relevant in vivo. It is therefore essential that protein-based structural studies are interpreted in the context of human samples and cell and animal models of disease. We describe here the biochemical techniques, monoclonal antibodies, cell biology, animal models, and stem cell technology that are useful to characterize the serpin polymers that form in vivo.

Unravelling the story of protein misfolding in diabetes mellitus.

Both environmental and genetic factors contribute to the development of diabetes mellitus and although monogenic disorders are rare, they offer unique insights into the fundamental biology underlying the disease. Mutations of the insulin gene or genes involved in the response to protein misfolding cause early onset diabetes. These have revealed an important role for endoplasmic reticulum stress in ß-cell survival. This form of cellular stress occurs when secretory proteins fail to fold efficiently. Of all the professional secretory cells we possess, ß-cells are the most sensitive to endoplasmic reticulum stress because of the large fluctuations in protein synthesis they face daily. Studies of endoplasmic reticulum stress signaling therefore offer the potential to identify new drug targets to treat diabetes.

Diabetes as a disease of endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress is an integral part of life for all professional secretory cells, but it has been studied to greatest depth in the pancreatic ß-cell. This reflects both the crucial role played by ER stress in the pathogenesis of diabetes and also the exquisite vulnerability of these cells to ER dysfunction. The adaptive cellular response to ER stress, the unfolded protein response, comprises mechanisms to both regulate new protein translation and a transcriptional program to allow adaptation to the stress. The core of this response is a triad of stress-sensing proteins: protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6. All three regulate portions of the transcriptional unfolded protein response, while PERK also attenuates protein synthesis during ER stress and IRE1 interacts directly with the c-Jun amino-terminal kinase stress kinase pathway. In this review we shall discuss these processes in detail, with emphasis given to their impact on diabetes and how recent findings indicate that ER stress may be responsible for the loss of ß-cell mass in the disease.