Establishing a large prospective clinical cohort in people with head and neck cancer as a biomedical resource: head and neck 5000

BACKGROUND: Head and neck cancer is an important cause of ill health. Survival appears to be improving but the reasons for this are unclear. They could include evolving aetiology, modifications in care, improvements in treatment or changes in lifestyle behaviour. Observational studies are required to explore survival trends and identify outcome predictors. METHODS: We are identifying people with a new diagnosis of head and neck cancer. We obtain consent that includes agreement to collect longitudinal data, store samples and record linkage. Prior to treatment we give participants three questionnaires on health and lifestyle, quality of life and sexual history. We collect blood and saliva samples, complete a clinical data capture form and request a formalin fixed tissue sample. At four and twelve months we complete further data capture forms and send participants further quality of life questionnaires. DISCUSSION: This large clinical cohort of people with head and neck cancer brings together clinical data, patient-reported outcomes and biological samples in a single co-ordinated resource for translational and prognostic research

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

The <i>VAChT^Y49N</i> mutation provides insecticide-resistance but perturbs evoked cholinergic neurotransmission in <i>Drosophila</i>

Global agriculture and the control of insect disease vectors have developed with a heavy reliance on insecticides. The increasing incidence of resistance, for virtually all insecticides, threatens both food supply and effective control of insect borne disease. CASPP ((5-chloro-1'-[(E)-3-(4-chlorophenyl)allyl]spiro[indoline-3,4'-piperidine]-1-yl}-(2-chloro-4-pyridyl)methanone)) compounds are a potential new class of neuroactive insecticide specifically targeting the Vesicular Acetylcholine Transporter (VAChT). Resistance to CASPP, under laboratory conditions, has been reported following either up-regulation of wildtype VAChT expression or the presence of a specific point mutation (VAChTY49N). However, the underlying mechanism of CASPP-resistance, together with the consequence to insect viability of achieving resistance, is unknown. In this study, we use electrophysiological characterisation of cholinergic release at Drosophila larval interneuron→motoneuron synapses to investigate the physiological implications of these two identified modes of CASPP resistance. We show that both VAChT up-regulation or the expression of VAChTY49N increases miniature (mini) release frequency. Mini frequency appears deterministic of CASPP activity. However, maintenance of SV release is not indicative of resistance in all cases. This is evidenced through expression of syntaxin or complexin mutants (sytx3-61/cpxSH1) that show similarly high mini release frequency but are not resistant to CASPP. The VAChTY49N mutation additionally disrupts action potential-evoked cholinergic release and fictive locomotor patterning through depletion of releasable synaptic vesicles. This observation suggests a functional trade-off for this point mutation, which is not seen when wildtype VAChT is up-regulated

The <it>Drosophila</it> nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

<p>Abstract</p> <p>Background</p> <p>Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect <it>Drosophila melanogaster</it> (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit.</p> <p>Results</p> <p>A full-length cDNA clone encoding the <it>Drosophila</it> nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in <it>Xenopus</it> oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [¹²⁵I]-α-bungarotoxin was detected in both <it>Drosophila</it> and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [¹²⁵I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments.</p> <p>Conclusion</p> <p>Data are presented demonstrating the ability of the <it>Drosophila</it> Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.</p

Central cholinergic synaptic vesicle loading obeys the set-point model inDrosophila

Experimental evidence shows that neurotransmitter release, from presynaptic terminals, can be regulated by altering transmitter load per synaptic vesicle (SV) and/or through change in the probability of vesicle release. The vesicular acetylcholine transporter (VAChT) loads acetylcholine into SVs at cholinergic synapses. We investigated how the VAChT affects SV content and release frequency at central synapses in Drosophila melanogaster by using an insecticidal compound, 5Cl-CASPP, to block VAChT and by transgenic overexpression of VAChT in cholinergic interneurons. Decreasing VAChT activity produces a decrease in spontaneous SV release with no change to quantal size and no decrease in the number of vesicles at the active zone. This suggests that many vesicles are lacking in neurotransmitter. Overexpression of VAChT leads to increased frequency of SV release, but again with no change in quantal size or vesicle number. This indicates that loading of central cholinergic SVs obeys the “set-point” model, rather than the “steady-state” model that better describes loading at the vertebrate neuromuscular junction. However, we show that expression of a VAChT polymorphism lacking one glutamine residue in a COOH-terminal polyQ domain leads to increased spontaneous SV release and increased quantal size. This effect spotlights the poly-glutamine domain as potentially being important for sensing the level of neurotransmitter in cholinergic SVs. </jats:p