Increased incidence of idiopathic paediatric facial palsy during the coronavirus disease 2019 pandemic.

BACKGROUND: Bell's palsy is a lower motor neurone facial weakness of unknown aetiology, although reactivation of a virus within the facial nerve has been proposed. METHODS: A prospective study was conducted of Bell's palsy cases presenting to our paediatric ENT unit over a 19-week period, from February to June 2020. Patients were invited for severe acute respiratory syndrome coronavirus-2 antibody testing. A text-message questionnaire was sent to other ENT centres to determine their observational experience. RESULTS: During the study period, 17 children presented with Bell's palsy, compared with only 3 children in the same time period in the previous year (p < 0.0001). Five patients underwent severe acute respiratory syndrome coronavirus-2 antibody testing, the results of which were all negative. Four out of 15 centres questioned perceived an increased incidence in paediatric Bell's palsy. CONCLUSION: Clinicians are encouraged to be vigilant to the increase in paediatric Bell's palsy seen during the coronavirus disease 2019 pandemic, which may represent a post-viral sequela of coronavirus disease 2019.

The negative impact of wearing personal protective equipment on communication during coronavirus disease 2019.

BACKGROUND: Coronavirus disease 2019 personal protective equipment has been reported to affect communication in healthcare settings. This study sought to identify those challenges experimentally. METHOD: Bamford-Kowal-Bench speech discrimination in noise performance of healthcare workers was tested under simulated background noise conditions from a variety of hospital environments. Candidates were assessed for ability to interpret speech with and without personal protective equipment, with both normal speech and raised voice. RESULTS: There was a significant difference in speech discrimination scores between normal and personal protective equipment wearing subjects in operating theatre simulated background noise levels (70 dB). CONCLUSION: Wearing personal protective equipment can impact communication in healthcare environments. Efforts should be made to remind staff about this burden and to seek alternative communication paradigms, particularly in operating theatre environments.

Coblation versus cold dissection in paediatric tonsillectomy: a systematic review and meta-analysis.

BACKGROUND: Cold dissection is the most commonly used tonsillectomy technique, with low post-operative haemorrhage rates. Coblation is an alternative technique that may cause less pain, but could have higher post-operative haemorrhage rates. OBJECTIVE: This study evaluated the peri-operative outcomes in paediatric tonsillectomy patients by comparing coblation and cold dissection techniques. METHODS: A systematic review was conducted of all comparative studies of paediatric coblation and cold dissection tonsillectomy, up to December 2018. Any studies with adults were excluded. Outcomes such as pain, operative time, and intra-operative, primary and secondary haemorrhages were recorded. RESULTS: Seven studies contributed to the summative outcome. Coblation tonsillectomy appeared to result in less pain, less intra-operative blood loss (p < 0.01) and a shorter operative time (p < 0.01). There was no significant difference between the two groups for post-operative haemorrhage (p > 0.05). CONCLUSION: The coblation tonsillectomy technique may offer better peri-operative outcomes when compared to cold dissection, and should therefore be offered in paediatric cases, before cold dissection tonsillectomy.

EGFRvIII-mediated transactivation of receptor tyrosine kinases in glioma: mechanism and therapeutic implications.

A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in significantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efficacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment.

A randomised comparison between an inexpensive, general-purpose headlight and a purpose-built surgical headlight on users' visual acuity and colour vision.

To determine if there are any differences in near visual acuity and colour vision between an inexpensive general-purpose light emitting diode (LED) headlight and a purpose-built surgical LED headlight. A prospective study was conducted sequentially comparing near visual acuity and colour vision, the headlights being tested in random order, in a testing room with a constant minimal amount of background light. The participants were NHS employee volunteers, with self-declared normal (or corrected) vision, working in occupations requiring full literacy. For visual acuity, outcome was measured by recording the smallest font legible when using each headlight when the subject read a near visual acuity test card. For colour vision, the outcome was passing or failing the Ishihara test. There was no statistically significant difference between the general-purpose and the purpose-built headlights in users' near visual acuity or colour vision.

Discovery of novel and potent benzhydryl-tropane trypanocides highly selective for Trypanosoma cruzi.

A benzhydryl tropinone oxime that is potently toxic to Trypanosoma cruzi has been previously identified. An SAR investigation determined that no part of the original compound was superfluous and all early SAR probes led to significant drops in activity. The only alteration that could be achieved without loss of activity was replacement of the aryl chloride substituent with chloro homologues. This led to the discovery of a trifluoromethyl-containing analogue with an EC(50) against T. cruzi of 30 nM and a cytotoxicity selectivity index of over 1000 relative to rat skeletal myoblast L-6 cells.

Radio opacity of various ENT foreign bodies in sheep's neck preparation.

We conducted an experimental study to evaluate the radio densities of different ENT foreign bodies. Various ENT foreign bodies were placed in the oesophagus of a sheep's neck preparation. An X-ray lateral view of the neck was taken, following the soft tissue neck protocol. Foreign bodies were grouped depending upon their radio densities and pixel value. The visibility of different materials on plain radiographs depends on their ability to absorb X-rays, and their inherent radio density and relation between it and the tissue in which they are embedded.

Unusual location for Castleman's disease.

OBJECTIVE: We present the first reported case of persistent, posterior triangle lymphadenopathy in a child, caused by Castleman's disease. CASE REPORT: A seven-year-old boy presented with a painless swelling in the posterior triangle of his left neck, with no compression of the surrounding structures. A histological diagnosis of Castleman's disease was made. Eventual treatment was by complete excision. At six-month follow up, there were no signs of recurrence. CONCLUSION: The causes of persistent cervical lymphadenopathy in children are many. Most are not significant, but some are life-threatening. Castleman's disease should be considered as a possible diagnosis in persistent childhood lymphadenopathy.

Etanercept, a tumour necrosis factor alpha receptor antagonist, and methotrexate in acute sensorineural hearing loss.

Patients with autoimmune inner-ear disease (AIED) are treated with high doses of steroids in the short term when suffering an acute hearing loss. As a consequence, substances such as methotrexate have been employed in the role of steroid-sparing agents. Additionally, it is known that tumour necrosis factor alpha (TNFalpha) is an important mediator of the inflammatory process, inhibition of which may be of benefit in AIED. This case report illustrates the use of a TNFalpha inhibitor in combination with methotrexate, which is known to be an effective combination in rheumatoid arthritis but has yet to be described for sensorineural hearing loss. We conclude that progressive AIED may respond well to TNFalpha inhibition, whilst more difficult cases, such as this example, could benefit from combining such therapy with methotrexate.

Audit of nasendoscope disinfection practice.

INTRODUCTION: Several options exist with regard to flexible pharyngo-laryngoscope sterilisation. We audited the use of disposable sheaths in our department over a six-month period. METHODS: A cost-analysis was performed and the advantages and disadvantages of this system were compared with several alternative options. RESULTS: We found that the overall cost of disposable sheaths averaged l4008 per month over a six-month period. We subsequently introduced chlorine dioxide (ClO2) wipes as a means of disinfection. Chlorine dioxide wipes have enabled a monthly saving of l3145 over sheath usage. Additionally, they meet health regulation requirements and are a convenient, cost-effective alternative to sheaths. DISCUSSION: The limiting factors, including time and financial issues, involved in nasendoscope disinfection are discussed. CONCLUSIONS: We have found chlorine dioxide wipes to be a satisfactory alternative means of nasendoscope disinfection. Possible time constraints aside, there are no advantages of sheath use over our current method. Chlorine dioxide wipes are also preferable from a financial point of view.

Substituted glycals as probes of glycosidase mechanisms.

D-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis beta-glucosidase in order to probe structure/function relationships in this enzyme. D-Glucal is shown to be a substrate (kcat = 2.3 min-1, Km = 0.85 mM) undergoing hydration with stereospecific protonation from the alpha-face to yield 2-deoxy-beta-D-glucose. 1-Methyl-D-glucal surprisingly serves as only a poor substrate (kcat = 0.056 min-1, Km = 57 mM), also undergoing protonation from the alpha-face. 2-Fluoro-D-glucal, however is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak (Ki = 24 mM) inhibitor. Similar behaviour was seen with almond beta-glucosidase and yeast alpha-glucosidase and for the interaction of 2-fluoro-D-galactal with Escherichia coli beta-galactosidase. A series of of alpha, beta-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis beta-glucosidase. Of these only 1-nitro-D-glucal functioned as a time dependent, irreversible inactivator (ki = 0.011 min-1, Ki = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed multiple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors (Ki = 3-96 mM).

Continuous, vesicle-based fluorimetric assays of 14- and 85-kDa phospholipases A2.

This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A2 (cPLA2) and the 14-kDa, human nonpancreatic, secreted phospholipase A2 (sPLA2). Phosphatidylcholines containing an arachidonyl chain at the sn-2 position and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at the sn-1 position were synthesized and shown to be substrates for cPLA2 in a fluorescence-based assay. Most of the assays make use of small and large unilamellar vesicles of substrate phospholipid, although the assay also works when the substrate is dispersed in Triton X-100 mixed-micelles. The cPLA2 assays can be carried out in a fixed time-point mode in which one of the products, the pyrene-containing lysophospholipid, is detected by rapid HPLC. Alternatively, the assay becomes continuous when bovine serum albumin is present in the aqueous phase; this protein extracts the pyrene-containing lysophospholipid from the vesicle, and this leads to the fluorescence of monomeric pyrene label. These assays are capable of detecting subnanogram amounts of cPLA2. The ester formed between gamma-linolenic acid and 7-hydroxycoumarin is also a substrate for cPLA2, and when incorporated into vesicles of the anionic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphomethanol, provides an assay in which the enzyme does not leave the vesicle surface (scooting mode). Unlike all of the previously reported, vesicle-based cPLA2 assays, a prolonged linear reaction progress is seen with the DOPM-based assay. An assay of sPLA2 with subnanogram sensitivity was developed which makes use of the substrate 1-palmitoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphomethanol and a lipid sink. The latter is composed of phosphatidylcholine vesicles, in excess of substrate vesicles, which do not bind sPLA2 but provide a trap for enzyme-produced 10-pyrenedecanoic acid. The fluorescence of monomeric pyrene label in sink vesicles is detected. A second sPLA2 assay using a single type of vesicle was developed based on the substrate 1,2-di(10-pyrenedecanoyl)-sn-glycero-3-phosphocholine present at 10 mol% in vesicles of the nonhydrolyzable anionic phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol. The action of sPLA2 on this fluorescent substrate leads to a separation of the pyrene chains resulting in fluorescence emission from monomeric pyrene. These cPLA2 and sPLA2 assays are ideal for inhibitor screening and analysis, and for studying the interfacial kinetics of these enzymes.

Probing the ionization state of substrate alpha-D-glucopyranosyl phosphate bound to glycogen phosphorylase b.

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505.

Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl-sn-glycero-3-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.

A continuous fluorescence-based assay for the human high-molecular-weight cytosolic phospholipase A2.

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl gamma-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA2 at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.

Identification of Cys139 and Glu207 as catalytically important groups in the active site of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyzes the antarafacial [1.3] allylic rearrangement of isopentenyl diphosphate (IPP) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). Active-site thiols at C138 and C139 were recently identified by covalent modification using active-site-directed irreversible inhibitors [Street, I. P., & Poulter, C. D. (1990) Biochemistry 29, 7531-7538; Lu, X. J., Christensen, D. J., & Poulter, C. D. (1992) Biochemistry 31, 9955-9960]. Kinetic studies were conducted with site-directed mutants of IPP isomerase (IPPIase) to evaluate the roles of these amino acids. C138S and C138V mutants were active catalysts with V/K values only 10-fold lower than that of wild-type IPPIase. In contrast, the C139S mutant was a poor catalyst, and the C139A and C139V mutants were inactive. Treatment of the C139S mutant with 3-(fluoromethyl)-3-butenyl diphosphate, an electrophilic active-site-directed irreversible inhibitor, resulted in inactivation of the enzyme by covalent modification of E207. The E207Q and E207V mutants were inactive, suggesting a role for the E207 carboxylate moiety in catalysis.

NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 complex. One mole of AACOCF3 per mole of enzyme is tightly bound in the active site while excess molar equivalents of the inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enzyme-inhibitor complex to be followed with 19F NMR. These results establish that the bound inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled enzyme-inhibitor complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the enzyme-bound inhibitor species is a charged hemiketal. These results are very similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine proteases, a structural model for the cPLA2.AACOCF3 complex is proposed.