It is clear that the field of organocatalysis is continuously expanding during the last decades. With increasing computational capacity and new techniques, computational methods have provided a more economic approach to explore different chemical systems. This review offers a broad yet concise overview of current state‐of‐the‐art studies that have employed novel strategies for catalyst design. The evolution of the all different theoretical approaches most commonly used within organocatalysis is discussed, from the traditional approach, manual‐driven, to the most recent one, machine‐driven. Computational approaches to organocatalysis: This review is focused on the different computational approaches that have been applied within the organocatalysis field. A huge effort from the theoretical chemistry field has been made in order to prove that de novo design of effective catalysts for different types of reactions is a long‐term target, yet a palpable phenomenon. Catalysis has been a paramount research topic in Chemistry since being separated from ‘alchemy’, proving its value by accommodating many important procedures in this age. Traditionally, it has been largely accepted that transition metal organometallic compounds and enzymes have been the two main types of effective catalysts, in particular in the field of asymmetric catalysts. Consequently, small organic molecules have been rarely used by synthetic chemists as catalysts during the past century, even though the very first pure organocatalyst was reported in 1912. Bredig described a modestly enantioselective alkaloid‐catalysed cyanohydrin synthesis  to study the possible pathways that may arise from a catalyst of dual nature such as chiral phosphoric acid catalyst, Figure Traditionally, distereovergence was explained only by sterical effects from the catalyst, however, mechanistic studies revealed that non‐covalent interactions (NCIs) were usually more important at guiding the reaction than mere sterical hindrance.ee) of a catalyst; showing how it is governed by the delicate balance between NCIs and the sterical factors present in the different transition states (TSs)involved.[Nowadays, computational chemistry is much more advance and the general workflow has changed over the last five years, Figure , bottom,involved.17, 18in silico determination of the intermediates along the current synthetic routes has led to a more profound understanding of the geometrical factors and NCIs that, individually or in a cooperative fashion, favour the production of the compound of interest. Overall, computational studies have revealed that the organocatalysts’ stereoselectivity is mainly determined by the presence (or absence) of electronic, steric and dispersion interactions between the catalyst and the species involved in reactivity ,R and S enantiomer were automatically analysed. Due to the numerous catalysts assessed through this automated methodology, a new origin of stereoselectivity was found and paved the way for the potential computational design of novel catalysts. Similarly in 2016, Doney et al. found new trends in the enantioselectivity of asymmetric propagations by screening a size‐able number of catalysts using AARON; leading to the design of a new catalyst with an ee exceeding 99 %.With a similar aim, the QM‐based open‐source Automated Reaction Optimiser for New Catalysts (AARON),Many other automatized software and packages for catalyst design are being gradually integrated into the field of organocatalysts; specifically the following programmes mentioned have the potential to be applied to the growing field. In 2018, Globally Optimal Catalysts, GOCAT,Despite the aforementioned advances in the automated design of novel catalysts, the description of the catalytic activity and enantioselectivity in organocatalysis often still requires expensive mechanistic QM analyses or MM approaches. Recently, many efforts have been dedicated to relating molecular properties – or descriptors – such as bond lengths, angles, vibrational modes and charges, with observable reactivity outputs, so as to quantitatively determine activities and selectivities of novel organocatalysts. Identification of patterns within these large datasets for subsequent interpolation becomes an impossible task, and one must rely on machine learning (ML) methods.ee values by Reid et al., by construction of a training set of MM and DFT‐derived quantitative structure‐reactivity relationships (QSRR) of 367 nucleophile additions to imines catalysed by aromatic phosphoric acids.ee values of similar catalytic processes within 5 %. A similar methodology was used for the determination of the DFT‐calculated ee value of asymmetric propargylation catalysts, whereby the construction of a dataset containing structural parameters from the intermediates preceding and succeeding the enantiodetermining TS resulted in an outstanding R2 value of 0.97 for out‐of‐sample catalysts.ee values within 3 % of the experimental.This was applied to the determination of The development of machine learning methods for finding and characterising novel asymmetric organocatalysts is gaining popularity given the myriad of readily available multi‐purpose software, such as PyTorch, Tensorflow or Keras. Furthermore, given the broad range of impact of organocatalysis, most current state‐of‐the‐art organocatalytic strategies opt to rely on variations of known, deeply studied routes. It has been discussed in the literature that novel pathways should not be derived from statistical models based on current models, but use template‐based strategies which allow for a more wide exploration of the field of action without the bias of current literature.With increasing computational capacity and new techniques, over the last two decades computational methods have provided a more economic approach to exploring different chemical systems. These developments have brought the synthetic and computational chemists together, and demonstrate the power of this synergy in catalyst design.In this review, we have summarised all the different computational approaches that have been applied within the organocatalysis community. Starting with the traditional approach in which computational models are used to elucidate the origins of selectivity in organocatalysis. Evolving to the so‐called, computational rational design, in which predictive models are generated involving quantum‐mechanical calculations on complex chemical systems; confirming the power of automatized methodologies in catalyst design; and finally, describing the use of thousands of chemical descriptors to elucidate patterns in multidimensional data.As it has been clearly stated in this review, a huge effort from the theoretical chemistry field has been made in order to prove that de novo design of effective catalysts for different types of reactions is a long‐term target, yet a palpable phenomenon.The authors declare no conflict of interest.1Nika Melnyk accomplished her Bachelor of Science degree in chemical sciences with medicinal chemistry from the Technological University Dublin (TUD) where she carried out her thesis on the computational analysis of antimalarials containing an endoperoxide bond under the supervision of Dr. Sarah Rawe and Dr. Cristina Trujillo. Her thesis established an interest for computational chemistry and she is currently doing her PhD under the supervision of Dr. Cristina Trujillo focusing on the catalysis of organic reactions through non‐covalent interactions in Trinity College Dublin.Iñigo Iribarren carried out his chemistry studies at ‘‘Universidad Autónoma de Madrid’’ as well as his master studies in theoretical and computational chemistry. He spent three months at ‘‘Sorbonne Universite’’ in Paris under the supervision of Dr Contreras developing a module for the non‐covalent interaction index software. He carried out his master thesis at ‘‘Instituto de Química Médica ’’ under the supervision of Prof. Alkorta focused on studying charged dimers linked by hydrogen bonds. Currently, Iñigo is at Trinity College Dublin doing his PhD under the supervision of Dr Cristina Trujillo focused on weak interactions on catalysis.Eric Mates‐Torres performed his undergraduate studies in the “Universitat Autònoma de Barcelona”, specialising in the computational study of organometallic catalysts under the supervision of Prof. Agustí Lledós and Prof. Jean‐Didier Maréchal. After this, he carried out his PhD in the group of Prof. Max García‐Melchor in Trinity College Dublin, focusing on the development of cathode materials for sustainable energy applications. He is currently collaborating with the group led by Dr Cristina Trujillo in Trinity College Dublin.Cristina Trujillo obtained her PhD in Theoretical and Computational Chemistry in 2008 at Universidad Autónoma de Madrid (UAM). During 2008–2016, she held several postdoctoral positions in Spain, Prague and Ireland. She joined Trinity College Dublin in 2019 being awarded with a Science Foundation of Ireland‐Starting Investigator Research Grant. She will join the University of Manchester as a lecturer in Computational Chemistry after summer. She has expertise in fundamental topics within Computational Organic Chemistry. Cristina's research group investigates the application of computational techniques in the design of organocatalysts, as well as the prediction and control of catalytic processes.


Developing an effective optogenetic strategy requires consideration of multiple factors, including the light‐sensitive protein that is used, the vector and method for gene delivery, and the target cell for expression because these in turn may affect the quality of vision that can be restored. Currently, at least four clinical trials are ongoing to investigate optogenetic therapies in patients, with the ultimate aim of reversing visual loss in end‐stage disease.Inherited retinal degenerations such as retinitis pigmentosa (RP) affect around one in 4000 people and are the leading cause of blindness in working age adults in several countries. In these typically monogenic conditions, there is progressive degeneration of photoreceptors; however, inner retinal neurons such as bipolar cells and ganglion cells remain largely structurally intact, even in end‐stage disease. Therapeutic approaches aiming to stimulate these residual cells, independent of the underlying genetic cause, could potentially restore visual function in patients with advanced vision loss, and benefit many more patients than therapies directed at the specific gene implicated in each disorder. One approach investigated for this purpose is that of optogenetics, a method of neuromodulation that utilises light to activate neurons engineered to ectopically express a light‐sensitive protein. Using gene therapy via adeno‐associated viral vectors, a range of photosensitive proteins have been expressed in remaining retinal cells in advanced retinal degeneration with  Abstract figure legend. Optogenetic approaches for vision restoration in end‐stage retinal degeneration. In inherited retinal degenerations, there is progressive loss of photoreceptors (rods and cones) but inner retinal structures remain largely intact. Optogenetic strategies aim to induce light sensitivity in remaining retinal cells using gene therapy, via ectopic expression of an opsin gene. The most widely investigated approach uses retinal transduction via an adeno‐associated virus injected either into the subretinal space or via intravitreal injection, with the aim of vision restoration . The inherited retinal degenerations are a heterogeneous group of disorders that result in photoreceptor cell dysfunction and cell death, leading to severe visual impairment. They affect around one in 4000 people and, with rare exceptions, there are no effective therapies  and this great heterogeneity has complicated attempts to develop effective treatment. Many different approaches have been tried, including the use of gene targeted therapies, neuroprotective agents, optogenetics, cell transplantation, stem cell therapy and retinal implants  is a rhodopsin with a microbial‐type chromophore that was originally isolated from the green alga 15 photons cm–2 s–1 required for ChR2 vs. 1010 photons cm–2 s–1 for cones and 106 photons cm–2 s–1 for rods) , which has improved light sensitivity and kinetics compared to ChR   is a light‐sensitive chloride ion pump derived from Rhodopsin and cone opsins are found natively in rod and cone photoceptors respectively and activation of each leads to cell hyperpolarisation. They are G‐protein coupled, and this has the significant advantage of permitting signal amplification. For example, a single activated rhodopsin interacts with more than 800 molecules of its G‐protein transducin, resulting in the closure of more than 200 ion channels in the photoreceptor outer segment disc membrane, from the absorption of one photon alone  and melanopsin (Opto‐mGluR6)  and rods with one . This multiplicity of connections allows for image processing and the creation of ON and OFF pathways, where a light stimulus excites one population of cells and inhibits another. These connections are further modified by horizontal cells to mediate contrast sensitivity. Cone ON bipolar cells synapse with ON ganglion cells, and OFF bipolar cells synapse with OFF ganglion cells, respectively. Rod ON bipolar cells synapse with amacrine cells, which in turn provide inhibitory input to OFF ganglion cells. There are at least 25 types of amacrine cell and more than 17 types of ganglion cell in the primate retina, with varying functions that facilitate our complex visual responses  to more closely mimic the retinal circuitry that is normally stimulated by light. RPE65) . These vectors have been used in human gene supplementation therapy clinical trials and in a Food and Drug Administration and European Medicines Agency approved retinal gene replacement therapy named voretigene neparvovec (Luxturna), for an early‐onset, severe retinal degeneration  residues to phenylalanine (F), can significantly improve the ability of AAV to penetrate the retina and increase transgene expression  and via intravitreal injection to target ganglion cells. Subretinal injection is surgically more challenging. It is performed in the operating theatre and requires intraocular surgery to remove the vitreous (vitrectomy), as well as penetration of the retina using a fine gauge needle followed by subretinal injection of the virus. There are associated small risks of retinal detachment, bleeding, infection , inflammation, raised intraocular pressure and developing cataract; however, this method allows for concentrated vector delivery to an area directly adjacent to the residual outer retina, and is the method of injection for the Food and Drug Administration approved Luxturna  have successfully targeted expression to ON bipolar cells , chicken beta actin promoter (A) and splice acceptor of the rabbit beta globin gene (G)], which has been demonstrated to facilitate effective transduction of the degenerate retina . The patients most suitable for these therapies are those with advanced visual loss  but with relatively intact inner retinal structures on optical coherence tomography (OCT) scans . A phase 1/2a trial by GenSight Biologics named PIONEER (NCT03326336) commenced recruitment in 2017, and reported outcomes from the first patient in 2021 , investigating intravitreal delivery of ChR2 using an AAV2 vector in 14 patients. Few data have been released regarding outcomes: no serious adverse effects were reported, although one of 14 subjects was documented to have vitreal cells and one out of 14 subjects was documented to have vitritis; however, it is not clear whether these outcomes were noted in the same patient . A fourth approach led by Nanoscope therapeutics investigates an intravitreal injection of an AAV2 based vector expressing a polychromatic opsin named multi‐characteristic opsin (MCO) alone, that is without the use of a retinal prosthetic device. A phase 1/2 dose escalation study of 11 patients has been completed (NCT04919473) and a press release on 1 year outcomes indicated an improvement in vision in all patients with regards to shape discrimination and mobility tests, with six out of seven patients in the high dose group reported to demonstrate gains in visual acuity . No safety concerns were reported. A phase 2 trial (NCT04945772) has now recruited 27 patients and further details of these studies are awaited.Another clinical trial of optogenetic therapy uses an alternative channel rhodopsin named ChronosFP in combination with a prosthetic device . Further details regarding the vector have not yet been released, but preliminary reports via a press release indicate that four patients were able to detect light and motion starting 2–3 months following treatment with ‘no safety concerns’ reported  and Fight for Sight (5099/5100).Peer Review HistoryClick here for additional data file.


Chest CT images of the patient in 2016, 2017, 2018, and 2019. The lesion was significantly larger compared with its size in 2016 A 62‐year‐old woman was diagnosed with pulmonary bullae accompanied by pulmonary nodule at a local hospital. The patient presented with an intermittent cough at the outpatient department of the Cancer Hospital Affiliated with Shandong First Medical University in 2016. Routine blood examination revealed slightly elevated neutrophil counts, no hepatitis B or hepatic function abnormalities, and negativity for carcinoma embryonic antigen and squamous cell carcinoma‐associated antigens.The expert multidisciplinary team (MDT) for pulmonary nodule at our hospital examined the computed tomography (CT) image from the other hospital Figure  and obseAspergillus ball”.In April 2020 Figure , the lesAspergillus infection is sometimes misdiagnosed as lung cancer. A nodule with a smooth margin in the pulmonary bullae is characteristic of a fungal ball formed by Aspergillus infection, and the lesion can enlarge slowly. At the initial diagnosis, this patient was correctly diagnosed by the expert MDT for pulmonary nodules at our hospital based on typical imaging manifestations, a positive GM test, and the relief of clinical symptoms after treatment with oral antibiotics.A fungal ball formed by In this case, the enlargement of the intrapulmonary nodule and “crawling out” of pulmonary bullae is rare imaging manifestations of fungal ball that have not been previously reported. This case report can provide a reference for the differential diagnosis of a fungal ball.


Ebstein's anomaly is an uncommon congenital malformation which might be associated with atrial septal defect and atrio‐fascicular Mahaim. Here, we report a known case of Ebstein's anomaly with atrial septal defect and concomitant atrio‐fascicular Mahaim pathway who underwent atrial septal defect device closure and concurrent ablation of accessory pathway. Careful paramedical examination is beneficial for patients by helping them to avoid undergoing an invasive treatment AFM is described as the presence of conducting pathways between AV node in RV and right bundle branch.ASD is another associated lesion with Ebstein's anomaly which needs attention. In these patients, the shunt is usually right to left, which increases with the age of the patient; the severity of the tricuspid regurgitation and right ventricular function. Closure of this defect can lead to increased central venous pressure and decreased cardiac output due to reduced left ventricular preload.In this paper, we report the case of a 32‐year‐old woman, a known case of Ebstein's anomaly with large ASD and concomitant atrio‐fascicular Mahaim pathway who successfully underwent ASD device closure with Cribriform Amplatzer device and ablation of accessory pathway.2A 32‐year‐old female, known case of Ebstein's anomaly and concurrent Atrial Septal Defect (ASD), referred to our center with complaint of frequent palpitation attacks. The patient was under medical treatment and regular follow‐up due to her stable condition. In basic electrocardiography, we observed normal sinus rhythm and no evidence of abnormalities.For better evaluation, three‐dimensional (3D) trans‐esophageal echocardiography (TEE) was done and revealed four secundum type ASDs in different levels of inter‐atrial septum (IAS) , with multiple small fenestrations that caused left to right shunt with appropriate rims for device closure  and also there was a large stretched patent foramen ovale (PFO) (0.3 cm*1.23 cm) with distance of about 1.1 cm from ASD.. In EPS, changes of electrocardiography (ECG) to left bundle branch bloke (LBBB) pattern during rapid atrial pacing with negative HV and the most fused AV and Mahaim potential in the postero‐lateral part of TV annulus area were observed. Furthermore, two different narrow QRS tachycardia were also induced; the first one was the atrial tachycardia (AT) from right postero‐septal area with cycle length of about 285 ms, HV and AH of about 45 and 115 ms, respectively, with retrograde atrial activation with earliest A in the HBE. The second was AT from infra‐crista region with cycle length of about 330 ms and HV and AH of about 45 and 115 ms, respectively, with retrograde atrial activation and with earliest A in high right atrium (HRA). Finally, EPS revealed that she had Mahaim accessory pathway (Atrio‐fascicular type) and Atrial tachycardia from postero‐septal and infra‐crista regions; all of them were successfully ablated. Moreover, because of atrial fibrillation (AF) rhythm, she received external electrical DC shock which fully converted AF rhythm to sinus rhythm.After successful ablation of accessory pathways, since morphological properties of her ASD made her suitable for device closure, she underwent diagnostic and therapeutic catheterization. During catheterization, hemodynamic study showed evidence of left to right shunt via ASD with ratio of pulmonary to systemic blood flow (QP/QS): 1.6. Patient hemodynamic and oximetry data is shown in Table 3Ebstein's anomaly is an uncommon congenital malformation with different presentationsIn our case, due to the history of palpitations and the possibility of preexcitation syndrome, at first, electrophysiological study was performed which demonstrated atrio‐fascicular Mahaim pathway in addition to atrial fibrillation and atrial tachycardia which is a relatively rare association in Ebstein's anomaly. Furthermore, in these patients, antiarrhythmic medication not only fails to effectively prevent tachycardia recurrences, but also carries a high potential for aggravating preexisting disturbances of physiological impulse generation or conduction and does not seem to be attractive to a mainly young population of patients requiring long‐term treatment. Hence, catheter ablation of cardiac arrhythmias particularly those related to presence of accessory pathways is the best choice for management of these patients.Another cardiac anomaly known to be associated with Ebstein's anomaly is ASD with the prevalence of 80%–94%.Today, trans‐catheter approach is the gold standard method for ASD closure in non‐Ebstein patients. For Ebstein patients who have traditionally been managed by surgical approaches, trans‐catheter closure of ASDs seems more attractive due to fewer associated complications including arrhythmias resulted from surgical scars.Based on 3D echocardiography finding, she had multiple fenestrated ASDs, and for closure of such ASDs, different strategies are available. Among them, we used Amplatzer Cribriform Multi‐Fenestrated Septal Occluder (Cribriform ASO) , which is a non‐self‐centering device that have two large equal sized discs with small linking waist diameter that can cover more than one adjacent defect.Another way for closure of fenestrated secundum ASD that accounts for 10% of all ASDs is simultaneous implantation of two or more devices or use of atrial septostomy with balloon and tear the strands and then placing a single larger atrial septal Occluder (ASO). Although these two methods have been successfully performed in some cases, they have side effects such as prolonged procedure time, risk of erosion and embolization, which are not seen when using Cribriform device.In general, we noticed concurrent presentation of Ebstein's anomaly with multiple small ASDs and a less common accessory pathway known as Mahaim accessory pathway. Beside these, we also challenged with a different kind of tachyarrhythmia known as Atrial tachycardia (AT).Our decision to perform this procedure was based on the patient's symptoms and the findings of echocardiography (appropriate rims and defect size) and angiography (significant left‐to‐right shunt) and in our patient, only a small postero‐superiorly ASD of about 2 mm remained, which had no hemodynamically significant residual shunt and we preferred to keep this defect as a backup because of the underlying patient's anatomy.This was the first time we tried to correct multiple ASDs with such an anatomy in a known case of Ebstein's anomaly by a cribriform device. There are few reports in the literature of trans catheter closure of the septum defect in Ebstein patients, which includes a small ASD with left to right shunt PFO.4Our experience with the reported case indicated that percutaneous device closure of multiple ASDs after percutaneous electrophysiological study is useful in management of patients even with complex anatomy and conductive pathway disorders and this should be as an available and minimally invasive approach on the table for management of these patients. It is worth mentioning that the important issue in these patients is the accurate anatomical and hemodynamic evaluation with different paraclinical methods. In general, timely decision and appropriate patient selection and type of intervention can help to reduce complications and improve the prognosis of patients.MA, ZK, and AF managed the patient. MA drafted the paper. IH and FM revised the draft. All authors read and approved final version of the paper.We took informed consent of the patient for reporting this case, additionally we ensured that her information would not be disclosed.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy


Cranial sutures join the many bones of the skull. They are therefore points of weakness and consequently subjected to the many mechanical stresses affecting the cranium. However, the way in which this impacts their morphological complexity remains unclear. We examine the intrinsic and extrinsic mechanisms of human sagittal sutures by quantifying the morphology from 107 individuals from archaeological populations spanning the Mesolithic to Middle ages, using standardized two‐dimensional photographs. Results show that the most important factor determining sutural complexity appears to be the position along the cranial vault from the junction with the coronal suture at its anterior‐most point to the junction with the lambdoid suture at its posterior‐most point. Conversely, factors such as age and lifeways show few trends in complexity, the most significant of which is a lower complexity in the sutures of Mesolithic individuals who consumed a tougher diet. The simple technique used in this study therefore allowed us to identify that, taken together, structural aspects play a more important role in defining the complexity of the human sagittal suture than extrinsic factors such as the mechanical forces imposed on the cranium by individuals' diet. The junctions between these bones (known as sutures) are composed of fibrous connective tissues, which, as a consequence of their greater elasticity compared to bone, enable a certain amount of movement between the individual units and therefore a level of flexibility in global cranial shape. From the initial stages of cranial development through to old age, sutures across the cranium achieve variable degrees of closure and do so at various stages of development, at times sealing completely leading to sutural obliteration. The sequence in which the various cranial sutures close is a well‐documented developmental process which can be used to estimate the biological age of individuals based on their bony remains . The morphology of this latter pattern is often described in the same terms used to describe wave functions (namely magnitude and frequency). Structurally, there are a number of ways in which the intricate morphology of sutures allows for several functions to be performed efficiently. Firstly, sinuosity will lock bones on either side into place, thereby counteracting potential shearing between them  is implicated in the structuring of cranial sutures. These strains vary as a direct consequence of changes in the masticatory regimes of mammals , to determine the impact of intrinsic  and extrinsic factors  on the morphological complexity of the human sagittal suture.2ossa coxae, where possible  of the suture was identified (point M) and marked with a pointed piece of modeling clay. Following this, the viewfinder of a Nikon D3100 camera was used to obtain a standardized orientation. The middle focus point was aligned with point M, and the extreme left and right focus points with the start and end points of the sutures . See Figure 2. All data relating to suture morphology were collected from this set of images.From an initially larger portfolio including all complete cranial vaults from all populations studied, 107 images (each depicting a unique cranium) were selected for inclusion within this study. Crania displaying substantial post mortem damage crossing the sagittal suture, as well as those with (near) complete obliteration of segments of the sagittal suture were excluded from the analysis. Furthermore, specimens with Wormian bones present at the junction of the sagittal and lambdoid sutures were only included when these bones took the form of small apical lambdoid ossicles, no larger than 2 cmBased on previous works, three indices were devised, thereby attempting to represent the sagittal suture morphology and complexity as thoroughly and simply as possible. The first was the “Complexity Index,” calculated by dividing the total length of the suture  by the linear distance from bregma to lambda (henceforth linear suture length). This is a variation on the most frequently used measure of suture complexity  and the nearby zygomatic arch (masseter), Rafferty and Herring  consistently achieve the highest levels of complexity (as measured by the complexity index and maximum extent), but older adults display the highest frequency of suture crossings (see Table In addition to the age of the individuals studied, the cultural context in which they lived also showed a significant relationship with complexity of the sagittal suture. Indeed, Mesolithic individuals display a generally lower suture complexity than those of other periods represented here (see Table Under the assumption of sutural complexity being dictated by the magnitudes of mechanical strains on the bone, the lower Mesolithic complexity observed here is surprising, as a decrease in dietary toughness would be expected to lead to a corresponding general decrease in sutural complexity of crania from the Neolithic and later periods Byron, , as wellAlthough changes in dietary habits within later archaeological periods appear not to have played a significant part in explaining suture morphology, extending the study to later populations would be valuable. The fast and intense industrialization of the past 250 years has had a profound role in the modification of human lifeways, including diet (Cordain et al., A number of studies have linked sutural complexity with dietary hardness in non‐human species (Burn et al., More sensitive morphology quantification methods such as surface scanning, and CT‐scanning can improve the performance of finer‐scaled studies. Additionally, the only suture investigated here is the sagittal suture, which runs along the superior edge of the cranial vault. However, mastication has been suggested to only exert small amounts of strain on the human vault (Behrents, Carlson, & Abdelnour, In addition, although the human skull develops along a general mammalian plan, growth of the cranial vault follows a unique trajectory to accommodate an early, rapid expansion of the brain. The human brain is large proportionally to body size when compared with that of other mammals and much of its growth occurs within the first 3 years of life (Peterson, Warf, & Schiff, Although this discussion of determinants of sagittal suture morphology has focused on various types of mechanical constraints, genetic factors should not be ruled out. Indeed, in forensic studies, the shape of the zygomaxillary suture has been utilized as an indicator of ancestry Gill, . Further5In this study, we investigated the morphological complexity of human sagittal sutures in a number of archaeological populations. Their cultural heterogeneity allowed us to assess the potential effect of differing masticatory regimes, such as that associated with the Mesolithic‐Neolithic transition, on sutural complexity. The decrease in cranial strains associated with the consumption of the softer, more heavily processed Neolithic diet appears to have resulted in an increase in the complexity index of the sagittal suture, contrary to the suggestions of previous studies. Nevertheless, this effect is quite small in comparison to that of intrinsic structural constraints associated with the various anatomical features of the human skull. However, the focus here was on the sagittal suture only. The application of the here‐described suture‐quantification methods on other cranial sutures in differing anatomical contexts will undoubtedly help toward the further clarification of the impact of both intrinsic and extrinsic factors on sutural complexity.Abigail Ash: Data curation; formal analysis; investigation; writing‐original draft; writing‐review & editing. Alexandra Anders: Resources; writing‐review & editing. János Dani: Resources; writing‐review & editing. László Domboróczki: Resources; writing‐review & editing. Eva Drozdova: Resources; writing‐review & editing. Michael Franken: Resources; writing‐review & editing. Marija Jovanovic: Resources; writing‐review & editing. Lidija Milasinovic: Resources; writing‐review & editing. Ildiko Pap: Resources; writing‐review & editing. Pál Raczky: Resources; writing‐review & editing. Maria Teschler‐Nicola: Resources; writing‐review & editing. Zdeněk Tvrdý: Resources; writing‐review & editing. Joachim Wahl: Resources; writing‐review & editing. Gunita Zariņa: Resources; writing‐review & editing. Ron Pinhasi: Conceptualization; funding acquisition; project administration; supervision; writing‐original draft; writing‐review & editing.The authors declare no potential conflict of interest.Table S1 Raw data used for this study.Click here for additional data file.Table S2 Results of the Mann–Whitney tests performed.Click here for additional data file.


The relationship between cartilage growth – mineralization patterns were studied in adult Rajidae with X‐ray morphology/morphometry, undecalcified resin‐embedded, heat‐deproteinated histology and scanning electron microscopy. Morphometry of the wing‐fins, nine central rays of the youngest and oldest specimens documented a significant decrement of radials mean length between inner, middle and outer zones, but without a regular progression along the ray. This suggests that single radial length growth is regulated in such a way to align inter‐radial joints parallel to the wing metapterygia curvature. Trans‐illumination and heat‐deproteination techniques showed polygonal and cylindrical morphotypes of tesserae, whose aligned pattern ranged from mono‐columnar, bi‐columnar, and multi‐columnar up to the crustal‐like layout. Histology of tessellated cartilage allowed to identify of zones of the incoming mineral deposition characterized by enhanced duplication rate of chondrocytes with the formation of isogenic groups, whose morphology and topography suggested a relationship with the impending formation of the radials calcified column. The morphotype and layout of radial tesserae were related to mechanical demands (stiffening) and the size/mass of the radial cartilage body. The cartilage calcification pattern of the batoids model shares several morphological features with tetrapods' endochondral ossification, that is, , but without the typical geometry of the metaphyseal growth plates.1. The wing‐fins system consists of stiff radials, mobile inter‐radial joints and a flat inter‐radial membrane adapted to the mechanical demand of wing wave movement.2. Growth occurs by forming a mixed calcified‐uncalcified cartilage texture, developing intrinsic tensional stresses documented by morphoanatomical data. (a) Bright field and (b) Phase‐contrast image. The cartilage growth pattern forms a mixed texture of calcified‐uncalcified matrix (not undergoing remodeling) which develops intrinsic, tensional stresses inside the tissue, histologically documented by elongation of chondrocyte lacunae and collagen fibers in the resilient uncalcified zones and by fractures in the neighboring calcified matrix. CharactIn endochondral ossification, cartilage is a temporary tissue before it is replaced by bone after the foetal and the metaphyseal growth phases, remaining later only on the surface of the joints during the whole lifetime. Otherwise, from tetrapods and birds, the cartilage of Chondrichthyes forms the definitive skeleton segments, which do not undergo secondary substitution (remodeling). The endoskeleton and fins cartilage of the latter has drawn deep interest in their phylogenesis  to analyze with X‐rays and trans‐illumination morphometry the wing‐fins radials growth pattern; 2) to compare the calcified layout of the wing‐fins, the pelvic girdle basipterigium fins radials and the tessellated endoskeleton segments; 3) we used a combination of light, scanning electron microscopy (SEM) and heat deproteination to study the mineral deposition progression of cartilage scaffold.This study has been carried out in the species 22.1Raja clavata Linnaeus, 1758, family Rajidae, class Chondrichthyes  and 5b, 5b (5 = oldest specimen), corresponding respectively to radials lines 1–4 and 5–8, while the external zones above bifurcation 1c, 5c corresponded to radials lines 10–13 . The number of rays and radials along each ray was determined in wing‐fins X‐rays, while for the pelvic basipterygia, only the number of rays was determined. In the right wing‐fin of the youngest and oldest specimen , the rays of the central sector, whose first radials line articulated with the mesopterygium and neopterygium . The difference in the mean analysis . The statistical analysis was carried out with the MedCalc program  using the Student Trans‐illumination allowed highlighting of the “tiles” calcified texture and layout of radials and pterygia.2.3Dry specimens (1 × 0.5 cm) of the wing‐fins radials and 1 mm thick transverse sections of the pterygia lying on glass slides were subjected to heat deproteination in a muffle furnace at 400°C for 24 h, then mounted with a cover slip and observed at higher magnification (40–200x) with an Olympus BX51 microscope .2.41). Specimens of radials and pterygia were decalcified in 10% ethylene‐diamine tetra‐acetic acid solution for 30 days and were embedded in paraffin blocks. Sections were stained with hematoxylin–eosin and mounted on glass slides. 2). Undecalcified specimens were embedded in Technovit 7200 resin , and 150–200 μm thick sections were prepared with the Exact cutting/grinding system  and stained with methylene blue‐acid fuchsine. 3). Single, heat‐deproteinated “tiles” were mounted on glass slides and observed under the light microscope.2.52critical point dried and secured on stabs with a bi‐adhesive tape. They were coated with a thin layer of gold in a vacuum sputter‐coater (Emitech) and examined with a Philips XL30 scanning electron microscope using secondary electron imaging (SEI) and backscattered electron imaging at 20 kV and 10 mm of working distance.Longitudinally sectioned surfaces of radials were dehydrated in increasing concentration of ethanol solutions, CO33.1The wing‐fins of the studied Rajiae were symmetric, showing an approximate triangular outline with two rounded lateral and posterior angles. The anterior wing skeleton ends with the sharpened propterygium tip, as shown by X‐rays, but not evident from the fish's outer look because the skin joins the head and rostrum together with the wings.We have counted 68 to70 rays in the wing fins of the studied specimens. This slight variation is explained by superimposition in X‐rays of the most anterior and posterior rays with the tip of the propterygium and the metapterygium. The single, total ray length and the number of radials in the ray increase progressively from the anterior extremity to the central sector to decrease in the posterior, rounded angle of the wing. The series of rays articulating with the mesopterygium and neopterygium  is the longest, with a higher number of radials and a perpendicular direction to the fish's long axis Figure . The len3.2a, b, and c and between the corresponding zones 1a, 1b, 1c and 5a, 5b, 5c of the youngest and oldest specimen  were examined with SEM in both the secondary and back‐scattered modes, documenting three calcified cartilage blocks intersected by the fracture plane but leaving undetected the others forming the joint basal disk above or below the latter plane. They were characterized by a high density of chondrocyte lacunae Figure . Outward4Gymnura marmorata and Urobatis halleri clearly showed a similar, ordered alignment of the inter‐radials joints parallel to the external outline of the wing, suggesting a specific phylogenetic character of the Rajidae family. These authors also reported inter‐species or intra‐individual variations of the mineralization pattern. In this study, shape differences between endoskeleton tesserae and radials cylindrical tiles were observed with a shape modulation in relation to the sectional area but without structural differences in the calcified cartilage. Maxwell et al.  of the wing‐fin were observed to begin as condensation of prechondrogenic mesenchyme later, undergoing segmentation at the joint sites  with a developmental mechanisms pattern shared by fins with the limbs of other vertebrates  to accomplish their mechanical function throughout the life cycle. The comparative analysis of radials length in the target specimens 1 and 5 allowed to examine the wing‐find growth in relation to age, the youngest (110 g) and the oldest fish (1250 g), respectively, documenting a significant decrement of radials mean length among the inner, middle and outer zones of the wing‐fin central sector. The distribution of inter‐radial joints showed a regular layout of curved rows parallel to the propterygium and the metapterygium, which suggests that the radials' length growth in the whole wing was set in such a way to maintain the ordered alignment of the joints. In the anterior and posterior wing‐fins sectors, the joint alignment is also achieved by reducing the radial number in the ray. This confirms a controlled radial length growth adapted to the mechanical demand. Schaefer and Summers  have repl et al.  documentTo the best of our knowledge, the microscopic trans‐illumination combined with the heat deproteination technique has not been so far applied to morphological and morphometric studies of cartilage calcification in the Rajidae wing‐fins. Our results have provided the following observations: 1) microstructure of the wing cylindrical tiles like that of the endoskeletal tesserae , resulting in calcium‐phosphate deposits in the matrix (Maroudas, The Rajidae cartilage growth of both wing‐fins and pelvic girdle metapterygia share similar patterns with the tetrapod's metaphyseal growth plate, such as chondrocytes duplication, volume increase and steering in rows where the intercellular matrix mineralization plays a driving role in cells orientation. Particularly in tetrapods' growth plate, this occurs between the calcified inter‐columnar septa, which orient the duplicating chondrocytes along the long bone axis, an action which is strengthened externally by the perichondral bone bark Dodds, . This stThe ultrastructural and developmental features of the tessellated endoskeleton of elasmobranchs have been extensively studied using synchrotron high‐resolution 3‐D techniques revealing in the course of development a complex lamellar, high‐mineral density layout with structures radiating outward, like spokes on a wheel, from the center of each tesserae to the contact with the neighbors (Seidel et al., Leucoraya erinacea with the definition “terminally differentiated in non‐hypertrophic state chondrocytes”. It can also be emphasized that the mass of the calcified matrix between these cells is scarce and less compact than that of the metaphyseal growth plate of tetrapods. In the same study, experimental cartilage injuries were produced by removing small wedges of tissue from the metapterygium, further providing evidence of a reparative chondrogenesis capacity. The “non‐hypertrophic state of chondrocytes” does not exclude the mineral deposition process based on H2O recall into the cell and concentration of Ca and PO4 ions in the intercellular matrix as suggested for the growth plate cartilage (Pazzaglia et al., 4 salts deposition into the collagen matrix network. The X‐ray imaging and the microscopic observations of heat‐deproteinated wing‐fins specimens provided so far unreported data on morphology and calcification in the radials tiles showing an evident correlation with the mechanics of the wing‐fins.To the best of our knowledge, few data have been so far provided on the cells and organic phase morphology in the calcified cartilage of elasmobranchs (Kemp & Westrin, To characterize and discriminate the taxa of the Mediterranean Rajidae, identification keys are currently used based on external and internal morphoanatomical traits (Serena et al., Ugo E. Pazzaglia: Conceptualization; data curation; investigation; methodology; supervision; writing – original draft; writing – review and editing. Marcella Reguzzoni: Conceptualization; funding acquisition; investigation; methodology; supervision; writing – original draft; writing – review and editing. Renata Manconi: Data curation; methodology; resources; validation; writing – review and editing. Piero Antonio Zecca: Data curation; funding acquisition; software; visualization. Guido Zarattini: Data curation; investigation; validation; visualization. Monica Campagnolo: Investigation; methodology; visualization. Mario Raspanti: Funding acquisition; project administration; validation.The authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this paper.


To examine the implementation of a physiotherapist-driven spirometry case finding service in primary care to identify new cases of COPD and confirm diagnosis of existing cases of COPD.1/FVC < 0.7). Outcome measures included number (%) of new cases of COPD, number (%) confirmed diagnosis of COPD and number (%) of high quality spirometry assessments with accurate interpretation.Four general practices were recruited. ‘At risk’ participants  and people with ‘existing’ COPD were identified from practice databases and invited to attend an assessment with a cardiorespiratory physiotherapist in each general practice. The physiotherapist performed pre/post-bronchodilator spirometry to identify or confirm a diagnosis of COPD (FEVn = 27). Quality of pre/post bronchodilator spirometries for FEV1 were classified as A (68%), B (19%) and C (5%).One hundred forty eight participants (mean age 70 years (SD 11.1), 57% female) attended a baseline assessment  from 748 people invited. Physiotherapists performed 145 pre/post bronchodilator spirometry assessments. Obstruction on post-bronchodilator spirometry was confirmed in 17% (19/114) of ‘at risk’ and 77% (24/31) of ‘existing’ COPD. Majority of cases were classified as GOLD Stage II (63%, Physiotherapists integrated into primary care performed high quality spirometry testing, successfully case finding ‘at risk’ patients and identifying potential misdiagnosis of obstruction in some ‘existing’ COPD cases.http://www.ANZCTR.org.au/ACTRN12619001127190.aspxANZCTR, ACTRN12619001127190. Registered 12 August 2019 – Retrospectively registered,  Desof < 0.7 , 19, 20.The Global Initiative for Obstructive Lung Disease (GOLD) guidelines advocate for active case finding of COPD in people with symptoms and/or the presence of risk factors . TherefoPhysiotherapists also contribute to COPD and chronic disease management, such as through the delivery of pulmonary rehabilitation programs and the use and interpretation of spirometry, yet the feasibility of a physiotherapist working in primary care to assist GPs with the diagnosis of COPD has not yet been examined. Considering physiotherapists’ wide skillset in both spirometry and chronic disease management, if successful, there is potential that physiotherapists embedded in primary care could also assist with early intervention where increased service delivery has the potential for substantial health benefit. Therefore, the primary aim of this paper was to examine the implementation of a physiotherapist-driven case finding service in primary care in order to determine if there is a role for physiotherapists in both identifying new cases of COPD and confirming diagnosis and severity in existing cases of COPD. A secondary aim was to examine the feasibility of this service in terms of clinic attendance and the quality of spirometry assessments completed by the physiotherapists with accurate interpretation.A pragmatic cross-sectional study embedded within a larger pilot study was conducted in Sydney, Australia. The study protocol was approved by the Northern Sydney Local Health District Human Research Ethics Committee  and was conducted in accordance with the WMA Declaration of Helsinki. The trial was registered with the Australian and New Zealand Clinical Trials Registry . The detailed methods of this study have been published previously . In brie1/FVC of < 0.7 [All potentially eligible participants were sent an invitation from the practice inviting them to take part in the study via letter or phone call. After obtaining written informed consent, participants were invited to attend a case finding appointment with the senior cardiorespiratory physiotherapist at the general practice. At the case finding appointment, participants completed baseline demographic questionnaires as well as the COPD Assessment Test (CAT)  and Modiof < 0.7 . Partici1 values and results are judged separately for pre-bronchodilator and post-bronchodilator results. A ‘grade A’ result constitutes at least 3 acceptable FEV1 and FVC manoeuvres with the difference between the two highest readings ≤ 0.150L. Grades B to E vary in number of acceptable manoeuvres and the variability in FEV1 and FVC readings. Grade U is classified as useable and grade F is not useable or acceptable [A member of the study team verified the physiotherapists’ interpretation of the spirometry results according to GOLD classification criteria. Quality of spirometry traces and results were judged according to the ATS/ERS acceptability and repeatability criteria and graded according to the repeatability grading system of quality A to F recommended by the ATS/ERS . The repceptable . ResultsThe primary outcome measures were the number (%) of new cases of COPD and number (%) of confirmed diagnosis of COPD and severity from previously diagnosed cases. Secondary outcomes consisted of the number (%) of eligible participants invited to attend, the number (%) that attended an appointment with the physiotherapist, the number (%) of spirometry assessments completed by the physiotherapist meeting ATS/ERS criteria , 36 withP values < 0.05 were considered statistically significant. Data was analysed using IBM SPSS Statistics for Windows, version 27.0. .All data are presented as mean ± standard deviation (SD) or number (%) unless otherwise specified for the baseline demographic data and primary and secondary outcomes. Statistical differences between groups at baseline were assessed using chi square tests of homogeneity or Fisher’s Exact tests  and independent t-tests (two-tailed) or Mann–Whitney U tests . n = 658 ‘at risk’ of COPD and n = 90 with an ‘existing’ COPD diagnosis) were invited to participate in the study. Of those invited, 40% (301/748) responded to the invitation and 21% (155/748) provided written informed consent to participate. A total of 148  participants attended a baseline appointment with the physiotherapist. The physiotherapist completed 145 pre and post bronchodilator spirometry assessments. Three participants were unable to perform spirometry due to poor technique from lack of understanding or the inability to perform despite multiple attempts and instruction. Of the spirometries completed, 114  participants were ‘at risk’ of COPD and 31  had an ‘existing’ COPD diagnosis. The physiotherapist correctly interpreted the level of obstruction according to GOLD classification criteria [A total of four GP practices were recruited and consented to participate in this study see Fig. . Particicriteria  in 98.6%n = 84) of the cohort were female. Participants in the ‘existing’ COPD group were significantly older (mean difference (95% CI) 7.9 years (3.7 to 12.2), p < 0.001) and had a significantly higher number of comorbidities (p < 0.001). The ‘existing’ COPD cohort also reported significantly higher CAT scores at baseline than the ‘at risk’ group (mean difference (95%CI) 6.9 (3.8 to 10.0), p < 0.001) and there was a statistically significant difference in mMRC scores between the ‘existing’ COPD group and ‘at risk’ group at baseline (p < 0.001).The baseline characteristics of the participants are presented in Table n = 43) of which 19 participants (44%) were part of the ‘at risk’ of COPD cohort. This indicates a case finding rate of 17% (19/114). The majority of participants were classified as GOLD stage II . Two participants from the ‘at risk’ group with airflow obstruction were referred by their GP for further follow-up testing for confirmation of diagnosis, of which one had airflow obstruction confirmed and one had a differential diagnosis of asthma. Only one participant in the ‘existing’ COPD group with no airflow obstruction on spirometry underwent further testing and was confirmed as a misdiagnosis.The results of the spirometry assessments are shown in Table The quality grading of spirometry assessments according to the ATS/ERS quality criteria for interpretation guidelines  is shownThe results of this study demonstrate that experienced senior cardiorespiratory physiotherapists embedded in a primary care practice to conduct case finding assessments via spirometry, are effective in identifying new cases of COPD. Our results also show that the senior cardiorespiratory physiotherapists were successful in interpreting lung function results for new cases of COPD as well as identifying potential misdiagnoses of COPD. To our knowledge, this is the first study to utilise physiotherapists working with GPs in primary care to assist with the identification of COPD.Our study resulted in a case finding rate of 17%, demonstrating that physiotherapists could be a successful option to integrate into the primary care team to conduct case finding for COPD. Previous studies looking at methods to improve early and accurate diagnosis of COPD within the primary care setting have identified undiagnosed cases, yet some have also reported logistical challenges which could present problems when integrating these methods into clinical practice , 38–40. n = 7). Of these, one participant underwent further testing and was confirmed as a misdiagnosis. There are many challenges to the diagnosis of COPD in primary care. Low levels of spirometry use is one such problem with a high proportion of patients with a diagnosis of COPD reported to have no spirometry results documented or have been diagnosed on the basis of non-spirometric criteria alone [Misdiagnosis and overdiagnosis of COPD remain ongoing issues in primary care , 20, 44 ia alone –47. Studia alone . An addiia alone . It is iParticipant response and uptake are important considerations in determining feasibility of screening programs and enabling effective translation into clinical practice. Overall attendance rate in our study was low with only 21% consenting to participate and 20% attending an appointment. However, this rate is similar to other case finding trials for COPD when using similar methods of recruitment , 50, 51.Screening completed by other health care professionals in alternative settings to primary care may also be a successful option to improve participation. For example, a pharmacy-based case finding service enabled pharmacists to screen for COPD then refer to GPs as appropriate which resulted in 92% of people approached completing the initial screening assessment . It is aThere were some limitations to this study. This was a feasibility study which only recruited a small number of people at risk of COPD from four general practices which limits the generalisability of findings. In addition, the practices were in a relatively affluent area of Sydney, Australia with generally low rates of smoking  so resulPhysiotherapists that are experienced and confident in the interpretation of spirometry could be useful in general practice to support GPs and practice nurses to improve the accuracy and interpretation of spirometry and diagnosis of COPD. The acceptability of this approach has not been explored and constitutes an important topic of future research. This will enable a deeper understanding of the effectiveness of this model as well as barriers and facilitators to implementing this model. It is important to establish if case finding through this approach is cost effective and can lead to early intervention and better health outcomes for patients which in turn, can potentially reduce the burden on healthcare systems. In addition, physiotherapists currently play a key role in management of COPD and other chronic diseases through delivery of pulmonary rehabilitation programs and exercise prescription, as well as secretion clearance techniques. Through integration of physiotherapists into the primary care team, it could provide them with the opportunity for discussions with patients surrounding physical activity and complex behaviour change. It remains to be seen if these discussions in a primary care setting can change behaviour and physical activity levels in people with COPD and lead to a change in disease trajectory.The results of this study suggest that experienced cardiorespiratory physiotherapists integrated into primary care can successfully perform and interpret spirometry, identify new cases of airflow obstruction, and assist with confirming diagnosis or misdiagnosis of ‘existing’ cases of COPD. Physiotherapists were able to identify a rate of case finding as well as a proportion of cases with an existing COPD diagnosis with no airflow obstruction that was similar to other studies. Further research is needed to determine if this model is cost effective and can lead to improved COPD management through early intervention.


The aganglionic bowel in short‐segment Hirschsprung's disease is characterized both by the absence of enteric ganglia and the presence of extrinsic thickened nerve bundles (TNBs). The relationship between the TNBs and the loss of enteric ganglia is unknown. Previous studies have described decreasing numbers of ganglia with increasing density of TNBs within the transition zone (TZ) between ganglionic and aganglionic gut, and there is some evidence of spatial contact between them in this region. To determine the cellular interactions involved, we have analysed the expression of perineurial markers of TNBs and enteric ganglionic markers for both neural cells and their ensheathing telocytes across four cranio‐caudal segments consisting of most proximal ganglionic to most distal aganglionic from pull‐through resected colon. We show that in the TZ, enteric ganglia are abnormal, being surrounded by perineurium cells characteristic of TNBs. Furthermore, short processes of ganglionic neurons extend caudally towards the aganglionic region, where telocytes in the TNB are located between the perineurium and nerve fibres into which they project telopodes. Thus, enteric ganglia within the TZ have abnormal structural characteristics, the cellular relationships of which are shared by the TNBs. These findings will help towards elucidation of the cellular mechanisms involved in the aetiology of Hirschsprung's disease. Glucose transporter 1 (GLUT1) is expressed in perineurial cells which as perineurium ensheath the endoneurium of extrinsic nerves.Enteric submucosal and myenteric ganglia in healthy intestine do not have a perineurium, but instead are ensheathed by a layer of telocytes, interstitial cells which form a cellular network,The region between the normal ganglionic and the aganglionic regions of the bowel in SS‐HSCR is characterized cranio‐caudally by a gradual loss of ganglia, and conversely by increasing numbers of TNBs.The histopathological characteristics applied in clinical practice to distinguish between the TZ and the normally innervated ganglionic region,22.1This study was carried out under the ethical approval obtained from the UK North West 3 Research Ethics Committee (Ref: 10/H1002/77). All samples were collected after informed consent from the patient's parent or guardian in keeping with the ethical approval. Human colon tissue was collected from 4 children with short‐segment Hirschsprung's disease  undergoing a pull‐through procedure and were aged between 1 and 5 months at time of surgery. Of the 4 patients, 3 children underwent primary pull‐through while 1 child had a stoma placed before undergoing pull‐through surgery. The pull‐through resected bowel was divided into 4 segments based on the macroscopic appearance of the rectosigmoid pull‐through resected tissue as well as the histopathology results of intraoperative biopsies. Full thickness samples were taken from segments identified as I‐IV, starting from the ganglionic region and through to the aganglionic region of the rectosigmoid pull‐through resected specimen Figure . Segment2.2The full thickness tissue specimens were fixed in 4% (w/v) paraformaldehyde for 4 h before being placed in 30% (w/v) sucrose for cryoprotection and embedded in Shandon Cryomatrix. The specimens were then cryosectioned at 7 μm and stored at −20°C.The following primary antibodies were used for immunofluorescence on serial sections Table : Hu, cal33.1To gain insight into the appearance of ENS and extrinsic TNBs across the resected tissue, we performed immunofluorescence for p75 which is expressed by both cells of the ENS and perineurial cells of the TNB,By combining immunofluorescence staining for the pan‐neuronal marker Tuj with p75, we confirmed that the structures in segments III and IV contained nerve fibres and were not enteric ganglia Figure , defininThese observations demonstrate the spatial context of the changes to the ENS through the resected tissue of SS‐HSCR colon by showing that ENS ganglia were lost, and extrinsic nerve bundles appeared which were ensheathed by a perineurium that co‐expressed GLUT1 and p75.3.2To determine the extent of the presence of ENS neuronal bodies in the neuronal structures displaying both ENS and TNB markers in segments II and III, we labelled for the neuronal marker Hu in combination with p75 Figure . Hu is o3.3We next asked whether the hybrid structures containing a perineurial sheath and Hu‐positive neuronal cell bodies, as observed in segment II, had characteristics of enteric ganglia. We addressed this by labelling for calretinin, a specific marker for a subset of ENS neuronsUsing confocal imaging, we analysed the calretinin distribution across the neuronal structures in segments I–III Figure . While eIt is important to note that calretinin is expressed in both the cell bodies and axons of a subset of enteric neurons.3.4To determine the extent to which the hybrid structures in the TZ have a normal telocyte ensheathment despite the presence of an abnormal perineurium, we co‐labelled sections across segments I–IV with antibodies for the telocyte marker CD34 Figure . Telocyt4In order to elucidate cellular relationships between the TNB and ENS, we have focussed on the transition zone between ganglionic and aganglionic regions of the HSCR bowel because previous observations have demonstrated that enteric ganglia and TNB co‐exist within the TZ.Our analysis of four cranio‐caudal segments along the transition zone of HSCR bowel confirms that the ENS and TNB undergo gradual reciprocal changes from ganglionic to aganglionic zones.While 3 of the 4 patients from which tissue was collected underwent pull‐through without prior surgery, one child had previously had a stoma placed before the pull‐through surgery from which the tissue was collected. It has been demonstrated in neonatal ratsDuring development, the perineurium of peripheral nerves is formed from specialized Schwann cellsIn order to understand the relevance of the hybrid structures, it is important to revisit the developmental process in which the ENS precursor cells migrate, populate and differentiate within the bowel. In the mouse, it has been shown that vagal neural crest cells colonize the myenteric plexus first and begin to differentiate into neurons before radial migration and projection of ENS precursor cells and Schwann cell precursor cells into the submucosal plexus.A hybrid‐like structure has been previously described in a colon biopsy by Kakita and colleagues,A detailed analysis of the four segments of HSCR pull‐through resected colon tissues using the neuronal markers calretinin, Hu and Tuj provided insight into the extent of intrinsic innervation across these tissues. Staining with calretinin visualized the change from cytoplasmic expression in the ENS neurons in the ganglionic bowel in segment I to a more granular localisation more distally (Segments II and III) before its absence in the aganglionic bowel in segment IV. While calretinin is only found in a subset of enteric neurons, nevertheless these data indicate, as previously hypothesised,It has been shown that in mouse models of HSCR, neurogenesis can occur from Schwann cell precursors (SCPs),Here, we have shown that telocytes ensheath the enteric ganglia in segments I and II, in line with their reported arrangement in normal enteric ganglia.Telocytes have a range of roles including the regulation of GI motility along with ICCsFurther analyses are required to determine the aetiology of the hybrid structures in the transition zone of HSCR, and the role of telocytes in ENS ganglia and in the perineurium of TNBs.Megan Smith: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft . Sumita Chhabra: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; validation ; visualization ; writing – original draft (supporting). Rajeev Shukla: Resources (supporting); writing – original draft (supporting). Simon Kenny: Funding acquisition (supporting); investigation (supporting); resources (supporting); supervision (supporting); validation (supporting); writing – original draft (supporting). Sarah Almond: Funding acquisition ; investigation (supporting); resources (supporting); supervision (supporting); validation (supporting); writing – original draft (supporting). David Edgar: Conceptualization ; formal analysis ; funding acquisition (supporting); investigation ; methodology (supporting); supervision ; validation ; writing – original draft .https://doi.org/10.1101/2022.06.15.22276240 but have not been otherwise published.The authors have no conflict of interest to report. The results presented in this paper are available in MedRxiv Appendix S1Click here for additional data file.


To evaluate differences in the cellular expression of epigenetic markers and oxidative stress in periodontitis lesions between current smokers and non‐smokers.Tobacco smoking is recognized as one of the major risk factors for periodontitis. However, the mechanisms by which smoking affects the progression of the disease remain to be determined.Twenty‐five current smokers and 21 non‐smokers with generalized severe periodontitis were included. From each patient, one soft tissue biopsy from a periodontitis site was harvested and prepared for histological analysis. The infiltrated connective tissue (ICT) was selected as the region of interest to assess the cellular expression of epigenetic markers and reactive oxygen/nitrogen species (RONS) by immunohistochemistry.Although the ICT of smokers and non‐smokers did not differ in size or in the expression of markers for DNA damage or oxidative stress, current smokers presented with significantly lower area proportions and densities of cells positive for the epigenetic markers DNMT1 and AcH3. In addition, periodontitis lesions in current smokers presented with a diminished antimicrobial activity, as indicated by significantly lower densities and area proportions of NOX2‐ and iNOS‐positive cells.Components of the host response and epigenetic mechanisms in periodontitis lesions in smokers are downregulated as opposed to lesions of non‐smokers. The study protocol was approved by the local human institutional review board of the University of Basel, Switzerland (EK: 159/06).Details on the recruitment of patients, inclusion/exclusion criteria and biopsy sampling procedures were previously reported by Schmidt et al.Before enrolment, all subjects received information about the study and signed an informed consent form. Clinical and radiographic examinations were performed. Salivary samples were used to measure cotinine levels to confirm the self‐reported tobacco use before initiation of the study .The patients were classified as generalized stage III or stage IV periodontitis according to the 2017 World Workshop on Classification on Periodontal and Peri‐implant Diseases and Conditions.2.1After dehydration and embedding in paraffin, the biopsies were stored at room temperature. From each tissue portion, 5 μm‐thick sections were produced using a microtome, dewaxed and incubated in DIVA antigen‐retrieval solution  at 60°C overnight. Following blocking of endogenous peroxidase and application of 4% bovine serum albumin (BSA), the sections were incubated with a primary antibody followed by incubation with Envision horseradish peroxidase (HRP)‐labeled polymer  for 30 min. Positive cells were detected using 3,3’ Diaminobenzidine (DAB) substrate (Agilent). The antibodies used for the immunohistochemical preparations and their dilutions are presented in Table 2.22) within the ROI.Qualitative and quantitative histological examinations of the inflammatory cell infiltrates were performed under a light microscope . Each section was captured with the Glissando Desktop Scanner  and transferred to a computer equipped with the computerized image analysis software Image‐Pro Premier . The analysis was performed by one trained investigator (C.D) who was blinded to the origin of the samples. The infiltrated connective tissue (ICT) was depicted and outlined with a mouse cursor as the region of interest (ROI). The smart segmentation tool of the IPP software was used to identify each cell marker, using a differential method analysis of color, intensity, morphology, and size. Thus, the total area occupied by positive cells was assessed for each marker and its percentage area relative to the total area of the ICT was calculated. In addition, the average cell size for each cell marker category was assessed. The number of positive cells in the ICT was computed using the data from the ROI total area, the average cell size, and the total area occupied by the positive cells. Cell numbers were expressed as total number and density of cells .The power calculation was based on assessments of cell phenotypes as reported previously.U‐test for independent variables. The null hypothesis was rejected at p < .05.Mean values and standard deviations were calculated for each variable, using the patient as the experimental unit. Differences between the two groups (current smokers and non‐smokers) were analyzed using the Mann–Whitney 3Micrographs of periodontitis sites obtained from a current smoker and a non‐smoker are presented in Figure 2 in samples from current smokers and 1.59 ± 0.27 mm2 in non‐smokers. The results from the histological analysis of the area proportions of positive cells are presented in Table The mean total area of the ICT was 1.47 ± 0.26 mm2) of positive cells within the ICT for each marker are listed in Table The results from the assessment of the total number and the relative density  or histone deacetylation (HDAC1/HDAC2), however, did not disclose any differences between the two groups. Histone acetylation and histone deacetylation are associated with several cellular processes, including gene transcription, and the relevance of analyzing markers of such functions in inflamed gingival tissues was demonstrated by Cantley et al.The present study failed to detect differences in proportions and densities of cellular markers for DNA damage (γ‐H2AX) and oxidative stress (8‐OHdG) between the two groups. Similar results were shown by Hendek et al.In addition to analysis of epigenetic and oxidative stress mechanisms, the present study also investigated cellular characteristics of antimicrobial activity in periodontitis lesions of the two groups of patients. Thus, periodontitis lesions in current smokers presented with lower densities of iNOS‐ and NOX2‐positive cells as opposed to periodontitis lesions in non‐smokers. As iNOS and NOX2 markers represent important functions of RONS of the innate host response, the findings in the present study indicate that periodontitis lesions of smokers exhibit a suppressed antimicrobial capacity. This observation is, however, in contrast with data presented by Özdemir et al.+, a precursor of NADPH, was noted in aggressive periodontitis sites than in chronic periodontitis sites.The finding in the present study on lower densities of NOX2‐positive cells in periodontitis lesions of current smokers indicates a disturbance in neutrophil functions, including the formation of neutrophil extracellular traps (NETs) and the elimination of pathogens through the “respiratory burst” process.Taken together, in addition to previous findings of fewer but wider vessels in periodontitis lesions of current smokers than in non‐smokers,In conclusion, while a clear linkage between epigenetics and inflammation remains to be established, the results of the present study point to an association between epigenetic modifications and severe periodontitis depending on whether or not the patients smoke tobacco products. These findings should be considered in risk assessments and classification of periodontitis patients. Clinicians need to be aware that smokers require specific attention in treatment planning and maintenance.AppendixS1‐S2Click here for additional data file.


The evidence base surrounding the transmission risk of ‘aerosol‐generating procedures’ has evolved primarily through quantification of aerosol concentrations during clinical practice. Consequently, infection prevention and control guidelines are undergoing continual reassessment. This mixed‐methods study aimed to explore the perceptions of practicing anaesthetists regarding aerosol‐generating procedures. An online survey was distributed to the Membership Engagement Group of the Royal College of Anaesthetists during November 2021. The survey included five clinical scenarios to identify the personal approach of respondents to precautions, their hospital's policies and the associated impact on healthcare provision. A purposive sample was selected for interviews to explore the reasoning behind their perceptions and behaviours in greater depth. A total of 333 survey responses were analysed quantitatively. Transcripts from 18 interviews were coded and analysed thematically. The sample was broadly representative of the UK anaesthetic workforce. Most respondents and their hospitals were aware of, supported and adhered to UK guidance. However, there were examples of substantial divergence from these guidelines at both individual and hospital level. For example, 40 (12%) requested respiratory protective equipment and 63 (20%) worked in hospitals that required it to be worn whilst performing tracheal intubation in SARS‐CoV‐2 negative patients. Additionally, 173 (52%) wore respiratory protective equipment whilst inserting supraglottic airway devices. Regarding the use of respiratory protective equipment and fallow times in the operating theatre: 305 (92%) perceived reduced efficiency; 376 (83%) perceived a negative impact on teamworking; 201 (64%) were worried about environmental impact; and 255 (77%) reported significant problems with communication. However, 269 (63%) felt the negative impacts of respiratory protection equipment were appropriately balanced against the risks of SARS‐CoV‐2 transmission. Attitudes were polarised about the prospect of moving away from using respiratory protective equipment. Participants' perceived risk from COVID‐19 correlated with concern regarding stepdown . Attitudes towards aerosol‐generating procedures and the need for respiratory protective equipment are evolving and this information can be used to inform strategies to facilitate successful adoption of revised guidelines. The predominant modes of disease transmission were thought to be via droplets >5 μm diameter and fomites. Airborne transmission was only considered to occur during medical interventions classified as aerosol‐generating procedures (AGP) .Our understanding of the viral dynamics and patterns of SARS‐CoV‐2 transmission has increased substantially over the course of the pandemic, including the recognition that airborne transmission occurs in the absence of AGPs , 5. EmerChanges to guidance may be welcomed by some healthcare workers but generate anxiety in others and this may lead to issues with rollout and implementation. The views of practicing anaesthetists towards existing AGP guidance and possible revision are unknown. This study aims to explore perceptions about the management of AGPs, attitudes to potential guideline alterations and consequent practice change.2This was a mixed‐methods study, comprising a survey disseminated to UK anaesthetists followed by qualitative interviews with purposefully selected survey respondents to explore their perceptions in greater depth. The Health Services Research Committee Executive Management Board of the Royal College of Anaesthetists reviewed the survey before circulation. Ethical approval for the qualitative component was granted by the University of Bristol Faculties of Life Sciences and Science Research Ethics Committee.https://www.kobotoolbox.org), was developed iteratively through two pilots. The initial draft was distributed to the members of the University of Bristol Anaesthetic Pain and Critical Care research group. A second iteration was distributed to the members of the Severn Trainee Anaesthetic Research Group. In each case, responses and feedback helped inform the final survey design. The final survey was distributed by the Royal College of Anaesthetists via email to their Membership Engagement Panel, a cohort of members at all career stages whose views contribute to the direction and strategy of the College via regular updates and surveys [The online survey, implemented using KoBoToolbox .Scenario 3: Tracheal intubation of the patient in scenario 2.Scenario 4: Supraglottic airway device insertion for the patient in scenario 2.Scenario 5: Tracheal intubation of a patient who had a negative SAR‐CoV‐2 polymerase chain reaction test, has self‐isolated for 14 days before admission and is asymptomatic for COVID‐19.https://www.R‐project.org). Thematic analysis was performed on comments from the free‐text sections that allowed respondents to provide additional commentary [Responses were screened for duplicates before analysis. Descriptive analysis was performed using R for respondent characteristics, Likert scale responses and response rates  to check for consistency in interpretation and coding; minimal semantic discrepancy was identified. The entire process of analysis was overseen by an experienced qualitative methodologist (LR), who met with the qualitative researcher (CO) regularly to discuss refinements to data collection processes and interpretation of data. The main qualitative researcher (CO) also kept a reflective diary to document personal reflections and experiences to mitigate against these inadvertently shaping data collection and analysis. We also sought to identify ‘negative cases’ and report these explicitly to ensure we reported the different dimensions of accounts. Member‐checking was undertaken to ensure clarification and understanding of interviewee responses. This was performed by summarising what was said both during and at the end of the interviews. Reporting of the qualitative elements of this study was guided by the consolidated criteria for reporting qualitative studies checklist.Survey results and qualitative findings are presented together, arranged according to the core topics investigated in the study; the emergent themes from the qualitative work are presented subsequently. The qualitative findings are supported by illustrative quotes  agreed to be contacted for the qualitative component of the study. In all, 18 interviews were undertaken between 31 December 2021 and 9 February 2022. The characteristics of all survey respondents and interviewees were compared with the 2020 RCoA Anaesthetic Workforce Census. This showed that our samples were broadly representative of the UK anaesthetic workforce across many domains  had received two doses of a COVID‐19 vaccine as compared with 91% of all NHS healthcare staff for the same period . FurtherRegarding tracheal intubation of an amber patient, 262 (84%) reported their hospital guidelines mandated the use of RPE, whereas it was the personal choice of 238 71%)  and 238 (71%), respectively. Regarding the risk of SARS‐CoV‐2 transmission 190 (57%) judged this to be higher for tracheal intubation of an amber patient as compared with speaking with the same patient pre‐operatively. For insertion of a supraglottic airway device in an amber patient, 201 (64%) of respondents' hospital guidelines advised, and 173 (52%) of respondents preferred the use of RPE. Removal of these devices in the operating theatre was recommended in hospital policies for 167 (83%) of respondents and 106 (53%) reported the need for fallow times following supraglottic airway device removal.3.1: “Am I rule follower or a rule breaker? I'm a rule follower, I think most of us aren't we at the bottom line” (Æ5).Of the respondents, 312 (94%) were aware of their hospital's IPC guidelines for AGPs and 264 (85%) felt this guidance prevented nosocomial SARS‐CoV‐2 transmission. Furthermore, 268 (86%) reported that guideline implementation added strain to working practices, yet 169 (63%) felt that this added strain was appropriately balanced against the risks of SARS‐CoV‐2 transmission .In agreement with the survey, some interviewees felt protected by RPE; however, others complied as they felt their clinical leadership (Æ8) role necessitated following their hospital policies. Others followed the guidance to avoid conflict within the workplace or to appease the fears of concerned team members , which has arisen from the pandemic and is difficult to challenge. Others suggested the disconnect between the static IPC guidance and the evolving pandemic has led to the observed variation in practice at both hospital and individual levels that was noted in the survey . Some also felt guidance did not account for important contextual issues, such as the introduction of rapid, sensitive screening techniques for SARS‐CoV‐2 and increasing rates of vaccination among staff and patients. These factors resulted in a loss of trust in the ‘IPC decision makers’ .Interviewees had divergent perspectives on the concept of AGPs. Tracheal intubation was the most contentious as to whether it was high risk and whether it should be classed as an AGP. Some interviewees believe tracheal intubation carries a higher risk of SARS‐CoV‐2 transmission than speaking to a patient pre‐operatively. However, many felt this increased risk was not from the procedure but from being near the patient, and their airway. Others felt administering neuromuscular blocking drugs reduced the risk of aerosol generation as the patient was then unable to breathe or cough .In the survey, 305 (92%) stated that the use of RPE and fallow times decreased operating list turnover, 376 (83%) felt RPE made team working more difficult and 255 (77%) felt it did not help communication. Furthermore, 202 (61%) stated RPE increased the risk of making clinical errors and 212 (64%) felt its use constituted an unacceptable environmental cost .The wearing of RPE was reported to negatively impact the development of patient rapport, communication with other team members and specifically to either increase the risk of, or directly contribute to, critical incidents .: “I follow generally what the hospital wants except that if I particularly feel that I'm wearing full PPE, that's what I'm doing. And if anyone wants to argue with me, they can do it”. (Æ18).The survey showed there was a bimodal pattern of perceived personal risk from COVID‐19 with distinct groups centred around low risk and high risk Fig. . YoungerThose anaesthetists with a higher perceived risk often had family members or friends who had experienced severe COVID‐19 symptoms  were comfortable or keen to move away from using RPE during airway management of an amber patient, but 168 (51%) remained anxious about the potential de‐escalation of IPC precautions, with 58 (17%) extremely anxious about this prospect. The degree of anxiety regarding de‐escalation had a positive correlation with perceived personal risk from COVID‐19; RR = 0.36, p < 0.001, 95%CI 0.26–0.45 .Many reported the pandemic and IPC guidelines had changed their practice. Some performed more ‘deep’ tracheal extubations and supraglottic airway device removals to reduce the risk of coughing, while others attempted to avoid AGPs entirely by using more regional or awake techniques. Supraglottic airway device use was more polarised with some avoiding their use as they felt the airway was not secure which was perceived to carry a higher risk of aerosol generation. However, others used supraglottic airway devices more frequently as their use has never been defined as an AGP. The increased use of videolaryngoscopy was frequently mentioned in both the free‐text comments and during interviews, and many interviewees stated they performed more rapid sequence inductions to avoid facemask ventilation .Underpinning perceptions of risk identified by the survey, many interviewees recounted the degree of fear and concern they felt at the start of the pandemic. A key theme to emerge from the qualitative interviews was the temporal changes in their personal perceptions of risk which generally reduced as the pandemic evolved .The impact of the COVID‐19 pandemic on training was frequently raised during interviews and in the survey free‐text comments. One trainee commented that many senior anaesthetists had altered their pre‐pandemic practice to avoid performing AGPs .Many interviewees felt the Royal College of Anaesthetists and the Association of Anaesthetists had not directly addressed the issues of AGPs despite IPC guidelines having a huge impact on routine anaesthetic practice. Interviewees reported a high level of trust in their professional bodies  and desired their contribution to and/or endorsement of any guideline changes that would impact practice (online Supporting Information, Table 4This study has demonstrated good support for the UKHSA IPC guidelines at national, hospital and personal levels during routine anaesthetic practice. Compliance with these guidelines has required dramatic changes to practice. Most respondents felt the IPC measures protected against occupational SARS‐CoV‐2 transmission and allowed patients to be treated without excessive fear for their own health. This survey has uncovered a broad range of perceptions regarding the guidelines and their implementation with substantial variations in practice, including significant deviations from the guidelines. This likely reflects the enormous challenge of implementing the AGP paradigm across the NHS during the pandemic. At the time of the survey, UK IPC policy recommended droplet PPE for all ‘non‐AGP’ patient interactions, irrespective of COVID‐19 status. Our findings indicate that many anaesthetists and their hospitals appear to disagree with this guidance, choosing and/or advocating RPE during pre‐operative assessments of patients with COVID‐19.Airborne precautions are not required for AGPs on patients/individuals if screening, triaging and testing have confirmed the absence of respiratory infection” [Many hospitals and anaesthetists have placed the emphasis of risk for SARS‐CoV‐2 airborne transmission on procedures, distorting risk assessments and management plans required for patient care interactions. The UK IPC guidance states “fection” . Yet, 40Over half of anaesthetists and their hospitals advocated RPE during supraglottic airway device use. Most felt insertion of a supraglottic airway device was a higher risk for SARS‐CoV‐2 transmission than performing a pre‐operative assessment in the same patient. These findings also support the previous report demonstrating 40% of UK hospitals are managing supraglottic airway device use as an AGP . AlthougThere is strong evidence that the implementation of UK IPC guidance has posed a challenge to the safe delivery of efficient healthcare resulting in decreased operating theatre utilisation and numbers of patients treated, impaired communication and teamworking, and increased stress and the risk of errors. There are concerns that the increased use of RPE is causing unacceptable and unwarranted environmental harm, as much of the equipment is single use and non‐recyclable. Another theme that emerged from the qualitative study is the impact on teaching and training of anaesthetists in terms of both reduced numbers of cases experienced and the issues of adequate communication while wearing RPE. There has yet to be a cost–benefit healthcare‐economic evaluation of the massive investment in RPE. The factors identified in this study should serve as a counterbalance to any assessment of the benefit from these infection control interventions.There is recognition that risk perception and personal beliefs have evolved since the first COVID‐19 wave, as understanding of the disease has evolved and new evidence has emerged. While most perceived that the risk associated with AGPs had decreased, the perceptions of some were fixed. This was particularly evident for anaesthetists who identified themselves at either end of the personal risk spectrum. Many expressed frustration that the AGP list and the associated IPC guidelines were not being updated as evidence accumulated during the pandemic. This was especially clear for the degree of aerosol generation from many procedures, the apparent lack of recognition of aerosol generation with coughing, the changed understanding of the different modes of transmission of SARS‐CoV‐2 and the impact of healthcare worker vaccination on the balance of risks.A substantial number of anaesthetists were keen to not wear RPE during tracheal intubation; however, there were at least as many who expressed a degree of anxiety regarding AGP precaution de‐escalation. Most felt there should be an allowance made for individual practice if anaesthetic procedures are to be removed from the UK AGP list. There was a strong desire for the professional bodies to have input into, or ratify, UK AGP guidance for anaesthetic procedures, as respondents felt these bodies carry considerable weight in shaping the opinions of anaesthetists and other healthcare professionals.The study has several limitations. The data represent the opinions of anaesthetists between November 2021 and February 2022, and we are aware there has been an evolution of opinions, beliefs and practices throughout the pandemic. The survey was conducted as the Omicron variant was beginning to circulate the UK, before its increased transmissibility, vaccine evasion and somewhat reduced virulence were understood. This was following a period of heightened concern regarding the Delta variant. The qualitative interviews were conducted during the period when Omicron BA.1 was the most common variant. All these factors may have had an impact on perception of COVID‐19 severity. We had a low survey response rate and this may reflect an element of survey fatigue and survey distribution during a late phase of the pandemic, yet respondent characteristics are representative of the UK anaesthetic workforce and the subsequent qualitative interviews have uncovered a wealth of information and insights that could not be determined by the survey alone. It is conceivable that anaesthetists with more polarised views were motivated to complete the survey and respond to the qualitative study invite. Nevertheless, it was those views that we sought to gauge the spectrum of opinions within the profession.Perceptions regarding AGPs and personal risk from COVID‐19 are evolving rapidly, as is the evidence around aerosol generation and the risks of transmission. The first major evidence‐based revision of a national AGP list for England  has receWe are not aware of any previous survey of anaesthetists' attitudes to AGPs; therefore, this study has provided important behavioural insights into the barriers, enablers and drivers that could facilitate successful communication and implementation of the new guidance. Any future guidance changes should have input from groups representing the clinical specialties affected, and ideally have endorsement by professional bodies. It is important that the group of anaesthetists that still identify as high risk, and those who are anxious about changes to practice, are supported through any phase of de‐escalation of airborne precautions, especially while the prevalence of COVID‐19 remains high in the community.Appendix S1. Original survey questions and link.Click here for additional data file.Table S1. Awareness and perceptions of aerosol‐generating procedure guidelines.Table S2. Impact of COVID aerosol‐generating procedure guidelines on practice.Table S3. Personal perception of risk.Table S4. Emergent qualitative themes.Click here for additional data file.