Our approach has proved to be cost‐effective and efficient in terms of time spent in the field against the data collected. The most demanding activities were the establishment of the transects and the vegetation sampling. The time spent downloading microclimatic data and testing seed germination was relatively short. Our sampling design allows: (i) to catch as much micro‐topographic variability as possible, both within and out of the tolerance range of the target species, (ii) to minimize the risk of recording identical micro‐topographic conditions compared with a random sampling scheme, and (iii) to ensure quick and relatively easy retrieval of the plots and the equipment both on a multi‐seasonal and multi‐annual basis.Extremely narrow endemic plant species (ENEs) are generally connected with microrefugia characterized by particular environmental conditions. In‐depth knowledge of the ecological requirements of ENEs is fundamental to plan appropriate conservation measures. Using cross‐cutting technology, this paper gives a multifaceted approach to collect on‐site data on the ecology of ENEs, defines the protocols for a correct sampling design and describes the type of equipment, the time and expenditure needed. Our sampling approach is based on two orthogonal transects, long enough to extrapolate the whole ecological gradient across the area of occupancy of the target species. Microclimatic data are recorded all along the transects through iButton technology, plus a weather station installed at the intersection of the transects. Microtopographic data are recorded with high‐resolution digital elevation model and sub‐metric GPS. Edaphic data are recorded along the transects through standard soil analyses and on‐site evaluation of the seasonal decomposition rate of organic matter. Additionally, vegetation sampling in 4 m Ptilostemon greuteri, we propose a cost‐effective, multifaceted approach utilizing cross‐cutting technology  to ensure the on‐site collection of data which can be used to: (i) interpret patterns of occupancy, (ii) monitor demographic variations, such as population numbers, size, density, and age structure as part of the assessment process of the target species, (iii) identify the most suitable sites for translocation, (iv) prepare the actions for its future conservation and (v) to ensure prompt reaction in case of changes that could compromise the survival of the species.Based on the experience gained from ongoing studies on a Sicilian strictly narrow endemic plant,  Its harsh morphology is shaped by the different responses of the outcropping rocks to the combined effects of past and ongoing karstic and tectonic processes. As a consequence, the whole mountain is furrowed by deep fluvial canyons, sinkholes, caves, and karrens .The climate of the study area is thermomediterranean  defined as: the vegetation patch with high percentage cover of the target species, nearest to the center of its area of occurrence  was used to record soil temperature and moisture. The iButtons were buried at a depth of 10 cm in the center of each plot along the transect, except for the CP, where a weather station , was installed in each of the two populations. The iButtons were attached to the metal pin marking the center of each plot in order to facilitate their retrieval. The weather station installed in the CP recorded rainfall, air temperature, soil moisture at two different depths (10–50 cm), and leaf wetness to quantify the effect of moisture condensation Figure .2.4Due to the steep ecological gradients occurring along the transects, we expected significant changes in the soil properties from the shaded plots toward those subject to long‐lasting direct solar radiation. Therefore, we collected substrate cores in each plot to perform standard physical, chemical and biological analyses. Details are reported in Appendix Additionally, we investigated the decomposition rate of organic matter using the Tea Bag Index . SketchUp is a 3D modeling software with a user‐friendly interface, that allows importing geo‐referenced DEM to reconstruct daily and seasonal variations of the shading/insolation in any spatial point with the plug‐in Shadow Analysis. As mentioned above, a sub‐metric GPS with maximum error smaller than the spatial resolution of the raster is fundamental to obtain accurate microtopographic data.To obtain precise micro‐topographic terrain data and to run the shadow analysis in SketchUp (DeltaCode®) environment, we used a DEM of 2 m spatial resolution provided by the Sicilian Regional Department of Territory and Environment .Our sampling design was adopted for three main aims: (i) to catch as much micro‐topographic variability as possible, both within and out of the tolerance range of the target species, (ii) to minimize the risk of recording identical micro‐topographic conditions compared with a random sampling scheme, (iii) to ensure quick and relatively easy retrieval of the plots and the equipment both on a multi‐seasonal and multi‐annual basis. It should be noted that we did not lose any equipment or data, apart from two teabags that were probably dug out by a porcupine ; funding acquisition (supporting); methodology (lead); writing – original draft (lead); writing – review and editing . Alessandro Silvestre Gristina: Conceptualization ; methodology ; writing – original draft ; writing – review and editing . Salvatore Pasta: Conceptualization (supporting); methodology (supporting); writing – review and editing . Giuseppe Garfì: Conceptualization (supporting); methodology (supporting); writing – review and editing . Leonardo Scuderi: Writing – review and editing (supporting). Laurence Fazan: Writing – review and editing . Viviane Perraudin: Writing – review and editing . Gregor Kozlowski: Funding acquisition (lead); writing – review and editing . Vito Armando Laudicina: Writing – review and editing . Roberto Venanzoni: Writing – review and editing (supporting). Riccardo Guarino: Conceptualization (supporting); methodology (supporting); writing – original draft ; writing – review and editing .The authors have no conflicts of interest to disclose.Appendix S1Click here for additional data file.


The simultaneous growth in our understanding of lymphoma biology and the burgeoning therapeutic options has come with a renewed drive for precision‐based approaches and how best to incorporate them into contemporary and future patient care. In the hunt for accurate and sensitive biomarkers, liquid biopsies, particularly circulating tumour DNA, have come to the forefront as a promising tool in multiple cancer types including lymphomas, with considerable implications for clinical practice. Liquid biopsy analyses could supplement existing tissue biopsies with distinct advantages including the minimally invasive nature and the ease with which it can be repeated during a patient's clinical journey. Circulating tumour DNA (ctDNA) analyses has been and continues to be evaluated across lymphoma subtypes with potential applications as a diagnostic, disease monitoring and treatment selection tool. To make the leap into the clinic, these assays must demonstrate accuracy, reliability and a quick turnaround to be employed in the real‐time clinical management of lymphoma patients. Here, we review the available ctDNA assays and discuss key practical and technical issues around improving sensitivity. We then focus on their potential roles in several lymphoma subtypes exemplified by recent studies and provide a glimpse of different features that can be analysed beyond ctDNA. Through these approaches, cataloguing of the genetic landscape for the majority of lymphoma subtypes is nearing completion.–7 In parThe term liquid biopsy was first used to describe methods to derive diagnostic information about a tumoural lesion from a blood sample. The term is now used in a broader sense to refer to the sampling and analysis of analytes from various biological fluids, most commonly blood but also urine, ascites, cerebrospinal and pleural fluid, all of which are relatively straightforward to sample compared to a traditional tissue biopsy, the historical gold standard for cancer diagnosis. There are various analytes of interest within these liquid biopsy compartments including circulating tumour cells (CTCs); cfDNA; circulating cell‐free RNA (cfRNA), nucleosomes, extracellular vesicles (EVs); tumour‐educated platelets (TEPs); proteins; and metabolites. As the Given the minimally invasive nature of liquid biopsies and the ability for tumour‐derived ctDNA to serve as a better representation of the patient's lymphoma, the evaluation of ctDNA in lymphomas has significantly expanded in recent years. In this review, we begin by summarising the rationale of ctDNA analyses, currently one of the most intensively studied analytes in liquid biopsy samples. We review some of the factors that can influence the validity and sensitivity of ctDNA assays and detail its clinical utility in the context of lymphomas, and end with an outlook of what is to come in the not‐too‐distant future.2Table One of the main barriers to rapidly incorporating precision strategies in lymphoma is the issue of having sufficient tissue to guide clinical decision‐making. Although pathological confirmation with a biopsy has been the gold standard in lymphoma and remains indispensable for diagnosis, there remain some notwithstanding challenges. Surgical excision biopsies are frequently touted in lymphoma guidelines as the ideal for diagnostic histological confirmation but in practice, radiologically guided core or needle biopsies are becoming more commonplace due to the ease and speed by which these can be arranged. Once obtained, a typical requisite is for the tissue to be fixed with formalin and embedded in paraffin in preparation for histological analyses. These two factors limit both the quantity and quality of tissue available for biomarker analyses after routine diagnostic assessments. Many lymphomas demonstrate biological heterogeneity, both spatially and temporally, 14, 15;3Figure Several factors can influence the sensitivity and, in turn, the clinical validity and utility of ctDNA‐based assays Figure 1. These3.12EDTA tubes, plasma isolation should be performed as soon as possible or at least within 4–6 h. The use of specialised cfDNA‐stabilising tubes (such as Streck cfDNA collection tubes) is preferable especially for multi‐centre studies as they extend the stability of cfDNA, allowing for processing to occur 2–7 days following collection. Additional care should be taken to optimise sample storage processes and avoid unnecessary freeze‐thaw cycles which hasten ctDNA degradation.A number of variables need to be accounted for in order to best optimise sampling, collection and processing of ctDNA from blood draws or other liquid compartments. First, ctDNA typically constitutes a small proportion of an individual's cfDNA, which in turn is also present at low concentrations. For cfDNA isolation from peripheral blood, sampling of at least 10 ml of blood is recommended to ensure an adequate amount of cfDNA is isolated. Second,3.2MYD88 L265P mutations highly recurrent in lymphoplasmacytic lymphoma or EZH2 Y641 mutations in diffuse large B‐cell and follicular lymphoma) and the assay has low multiplexing capabilities. In contrast, NGS allows for massively parallel sequencing of a large number of genetic loci simultaneously. As the maximum number of sequenceable base pairs is limited by the current NGS technology, increasing genomic coverage results in a decrease in the depth of sequencing achievable. One of the initial NGS‐based assay used in B‐cell lymphomas takes advantage of the unique biology of B‐ cell receptors. As each lymphoma comprises specific clonotypes defined by immunoglobulin VDJ rearrangements, these can be identified in lymphoma tissue through high throughput sequencing using universal primer sets  to encompass all possible rearrangements of the immunoglobulin loci. These pre‐determined tumour‐defined clonotypes are then trackable in the ctDNA of the patient. Due to the high sequencing depth required to accurately call low allele frequency variants, whole exome (WES) or genome sequencing (WGS) strategies run the risk of missing low‐frequency variants and therefore high depth sequencing of targeted panels of genes known to be recurrently mutated in lymphoma is more the norm. This is the basis of the cancer personalised profiling by deep sequencing (CAPP‐seq) approach that employs a comprehensive targeted gene panel design to detect lymphoma‐associated single nucleotide variants (SNVs), copy number alterations (CNAs) and structural variants (SVs)[Given the potential low frequency of putative somatic gene variants against elevated levels of background noise, high sensitivity is a requisite for ctDNA assays. There are primarily two genotyping methods currently in use: polymerase chain reaction (PCR)‐based detection methods such as droplet digital PCR (ddPCR) and next‐generation sequencing (NGS)‐based methods. ddPCR offers high sensitivity (VAF ≤0.01%), is relatively straightforward to set up and has a quick turnaround without the need for complex bioinformatics analyses. However, as amplicons are limited to less than 1 kb , their utility is limited to the detection of known recurrent “hotspot mutations” (such as nts (SVs). There a3.3tumour‐informed approach. In cases where paired tumour tissue is unavailable, a tumour‐agnostic approach where ctDNA analysis is performed without prior knowledge of a patient's tumour mutation profile can be employed. The advantages of the tumour‐agnostic approach are that profiling is less complex, associated with lower costs, faster turnaround time, and the ability to detect emerging resistant mutations. However, the trade‐off in the absence of the tumour genotype is a higher rate of false positives and the inability to track a known tumour VDJ clonotype. Though more resource‐intense, a tumour‐informed approach aids in identifying false negatives in ctDNA and is thus particularly well‐suited for broader minimal residual disease (MRD) detection  and disease recurrence monitoring.The ideal scenario is one where adequate tumour tissue is readily available in all patients so that the somatic variants are identified prior to or alongside ctDNA profiling to allow the identification of baseline tumour genotypes and a reference for bespoke longitudinal ctDNA surveillance, a so‐called Another consideration is the inclusion of germline DNA such as derived from saliva or buccal swabs to identify variants that are truly somatic in nature. As normal haematopoietic cells accumulate somatic mutations during ageing which can drive clonal expansions of haematopoietic cells, referred to as clonal haematopoiesis (CH), 29 whic44.133 and although approximately 50% of patients can be cured with induction treatment alone, patients who relapse or are refractory to salvage or autologous stem cell transplantation have a very poor prognosis with 2‐year overall survival from relapse of around 20%[Diffuse large B‐cell lymphoma (DLBCL) is the most common high‐grade lymphoma and exhibits marked biological and clinical heterogeneity. Over 20round 20%. Whilst round 20%, 41, 42 round 20%, 44 and round 20%, 46.p < 0.0001)[Among all lymphoma subtypes, applications of liquid biopsy have been best examined in DLBCL. The rarity of circulating tumour cells in aggressive lymphomas has sparked the investigation of additional liquid biopsy analytes, principally ctDNA in DLBCL. Both plasma‐derived and serum‐derived cfDNA has been studied in DLBCL and although higher concentrations of total DNA can be isolated from serum samples, cfDNA is contaminated by high fragment length genomic DNA released by lysing cells during in vitro sample preparation and thus plasma has become the specimen of choice for peripheral blood ctDNA analysis. Initial< 0.0001). Additio< 0.0001), 50. Wit< 0.0001).These studies convincingly demonstrate a plausible role for ctDNA in follow up and post‐treatment MRD monitoring and risk stratification in DLBCL. A rather fascinating study by Kurtz and colleagues highlights the potential of an approach utilising dynamic risk assessment whereby a continuous individualised risk index (CIRI) based on a Bayesian statistical model is calculated by considering both pre‐treatment and dynamic on‐treatment risk factors in order to estimate outcomes at any given time point during a patient's disease or treatment journey. How theKMT2D and TP53 mutations at progression[The earliest studies investigating ctDNA in DLBCL established that plasma ctDNA could serve as a good surrogate to tumour biopsies with a high concordance between the genotype of diagnostic biopsies and plasma ctDNA. This is relevant given the challenges and infrequency of repeat biopsies, especially in the context of relapsed or refractory DLBCL (rrDLBCL). By performing targeted gene panel sequencing of primarily the plasma ctDNA in over 100 patients with rrDLBCL, Rushton and colleagues demonstrated differences in the mutational profiles between diagnostic and rrDLBCLs, with enrichment in ogression. It alsoogression.EZH2 Y641N and MYD88 L265P may have utility in identifying those who will benefit from specific therapies and in monitoring responses[Given the broadening gamut of therapies, particularly in the rrDLBCL setting, predictive biomarkers and response assessment using ctDNA is an important biomarker opportunity. Droplet digital PCR for tracking a number of recurrent actionable hotspot mutations such as responses, 57, altresponses, 41, 42 responses. Crucialresponses, 60.4.2Cancers that affect the CNS pose a particular challenge because of the invasiveness and difficulty in safely accessing tissue for diagnosis. Due to anatomical proximity, ctDNA can be more readily detected in CSF‐derived cfDNA from CNS lymphomas, primary and secondary brain cancers, 62 thanMYD88 L265P mutation due to its presence in greater than 80% of PCNSL tumours. MYD88 mutations are exceptionally rare in non‐lymphoma CNS malignancies and thus could be used to distinguish from PCNSL. The sensitivity in detecting MYD88 mutations in plasma‐derived ctDNA has varied in different series. In one study, MYD88 mutations were identified in approximately one‐third of PCNSL cases that harboured the mutation within the tumour sample, with considerably lower variant allelic fractions (VAFs) in the ctDNA compared to tumour DNA[MYD88 L265P mutations were detectable in CSF from 20/26 cases of CNS lymphoma[The earliest studies evaluating the utility of ctDNA focussed on the umour DNA; althougumour DNA. Watanab lymphoma suggestin = 6), one case of systemic DLBCL with subsequent SCNSL involvement but in no cases of DLBCL without CNS involvement (n = 12)[BTG2, PIM1, DUSP2, ETV6 and CXCR4) were associated with high‐risk CNS IPI scores[Secondary CNS lymphoma (SCNSL) is a rare but devastating complication of systemic DLBCL and as such, there have been numerous efforts to identify patients with a low and high risk of CNS relapse in order to rationalise the use of toxic CNS‐penetrating chemotherapy regimen. The majority of CNS relapse prognostication tools rely on clinical parameters such as the CNS‐IPI. In a re (n = 12) indicatiPI scores. These p4.3CD79B and MYD88) but also showing recurrent rearrangements in the checkpoint ligands PD‐L1/PD‐L272.Intravascular large B‐cell lymphoma (IVLBCL) is a rare subtype of extranodal aggressive B‐cell lymphoma that is peculiarly confined, although not exclusively, to the lumen of blood vessels (intravascular space) without lymphadenopathy, making diagnosis extremely challenging. Due to the diffuse nature of the lymphoma infiltrate and lack of tumour masses, IVLBCLs are difficult to detect with conventional imaging and are often missed by tissue biopsy leading to delays in diagnosis and poor patient outcomes, highlighting an opportunity for ctDNA analyses to aid diagnoses. Recent data suggest that ctDNA levels may be higher in IVLBCL compared to DLBCL. In a se4.4n = 31). However, some patients did go on to relapse at a later stage indicating that, at least in this study, ctDNA genotyping is highly specific but lacks sufficient sensitivity for use as an MRD tool although this could in part have been due to the limited gene panel and assay sensitivity.A detailed understanding of the genomic landscape in classical Hodgkin lymphoma (cHL) had lagged behind other B cell lymphomas owing to the rarity of Reed‐Sternberg cells (1–2%) within cHL biopsies, 75, 76.In a prior study with a 77‐gene CAPP‐seq based targeted panel, the authors demonstrated a high concordance between variants present in ctDNA and tumour DNA (nearly 90%). In a cohort of 24 advanced‐stage cHL patients, a 2‐log fold reduction in ctDNA levels following two cycles of ABVD chemotherapy was associated with a PFS and overall survival advantage, finding4.5Follicular lymphoma (FL) is the most common indolent NHL with heterogeneity in clinical behaviour. Early‐stage FL can achieve long remissions in about half of the patients with standard treatment modalities. For advanced‐stage FL, a subset can initially be managed by active monitoring, others may require multiple lines of therapy and there remains a high‐risk group who relapse early or transform to DLBCL and have significantly inferior outcomes. AccuratIn an initial series of 34 patients from the PRIMA trial, using clonoSEQ, VDJ clonotypes were identified in the plasma of 74% of patients, with a high ctDNA level at diagnoses corresponding with a significantly shorter PFS providinEZH2 mutations detected by ddPCR in ctDNA, the EZH2 VAFs fluctuated in accordance with tumour burden and in response to immunochemotherapy[The mutational landscape of FL, like many other lymphomas, is well defined, 84, 85 motherapy. This mamotherapy.The capability to predict FL transformation is an area of interest due to adverse outcomes. In an early pilot series, transformation‐specific mutations within the tumour could be identified in the plasma ctDNA several months before the onset of clinical transformation, pointin4.6There have been small but insightful studies in other B‐ and T‐cell lymphoma subtypes. In mantle cell lymphoma (MCL), Agarwal and colleagues showed that ctDNA genotyping results were non‐inferior to traditional flow cytometry or allele‐specific PCR MRD detection methods. Mutations in genes related to chromatin remodelling were associated with treatment resistance to ibrutinib and venetoclax and could easily be tracked in plasma ctDNA samples.There is likely to be a tremendous benefit in T‐cell lymphomas as these subtypes more commonly are associated with extra‐nodal disease, which can at times be challenging to re‐biopsy. Plasma EBV‐DNA measurement has been used in post‐transplant lymphoproliferative disorder (PTLD) and extranodal NK‐T cell lymphoma (ENKTCL) as a diagnostic and disease‐monitoring tool. In ENKTKMT2D were associated with advanced stage and higher tumour metabolic volume[In a series of 65 patients with ENKTCL, there was over 90% concordance between plasma‐derived ctDNA with tumour tissue genotyping and certain mutated genes including ic volume. Anotheric volume. These sCompared to B‐cell lymphomas, studies evaluating the feasibility of ctDNA analyses in other T cell lymphomas have thus far been scarce and in small cohorts, primarily of angioimmunoblastic and other peripheral T‐cell lymphomas, 94. Man5Figure Broad applicability: Larger studies will be required to assess the feasibility and suitability of these assays across the lymphoma subtypes.Dual prediction and prognostication: In addition to comparisons with standard response assessment tools like imaging, the next steps would entail investigating whether these tools have sufficient predictive and prognostic accuracy to inform de‐escalation or escalation of therapy (for example maintenance or consolidation) as well as the selection of novel therapeutic strategies.Molecular‐based endpoints: Can we envisage a future of defining remission molecularly in lymphoma? This would require the development and evaluation of more sensitive liquid biopsy assays to measure and monitor MRD.Approved ctDNA‐based assays: For the clinical adoption of ctDNA assays, a detailed roadmap from careful evaluation and standardisation of the processes and methodologies to establishing utility, reproducibility and cost‐effectiveness is required.The potential clinical applications of ctDNA range from aiding diagnoses to treatment selection and response and progression monitoring Figure 2. Altog6Aside from quantifying and genotyping ctDNA, there is an emerging multitude of alternative ctDNA and non‐ctDNA parameters through which to derive additional information about the underlying tumour.Current ctDNA profiling approaches focus on the high depth sequencing of a single locus or well‐defined targeted gene panel analyses. However, there are limitations to this strategy especially in the setting of evaluating low burden disease settings such as MRD that is of relevance in lymphomas. As the tumour burden is significantly lower at MRD states, there is a ceiling beyond which a higher depth of sequencing and complex bioinformatics analyses will not achieve the necessary resolution and sensitivity due to the low number of tumour‐specific DNA molecules in this context. Alternative approaches are being explored, whereby broader genome‐wide profiling using whole‐genome sequencing coupled with machine learning to track multiple genetic aberrations from mutations to copy number alterations at much lower sequencing depths could mitigate some of the challenges of restricted gene panel sequencing.Beyond genetic changes, epigenetic deregulation is now a recognised hallmark of both B‐ and T‐cell lymphomas. DNA methylation of CpG sites is an important epigenetic regulator of gene expression with different tumour types exhibiting specific methylation signatures. Tumour‐specific methylation signatures can be detected in cfDNA and have been used to trace the tissue of origin of cfDNA or indeed to directly detect cancers with remarkable sensitivity and specificity, 97. UsiAnother evaluable and informative feature of ctDNA is the pattern of cfDNA length and fragmentation. It is known that cfDNA fragments roughly correspond to the length of DNA wrapped around a nucleosome (∼147 bp) plus linker DNA. Tumour derived ctDNA fragments have been shown to have greater variability and typically are distinctly shorter than non‐tumour derived cfDNA, in the region of 90–150 bp, 102. ThAs cfDNA is predominantly released by apoptotic cells, it circulates in the form of nucleosome‐protected DNA fragments in the circulation. As nucleosome positioning is an epigenetic determinant of gene expression that is cell or tissue specific, studies have demonstrated that gene expression could be inferred from the differences in the nucleosome patterns between highly versus lowly/silently expressed genes (detected as differences in the sequencing depth at transcription start sites upstream of the genes) and this in turn could be used to determine the cell or tissue‐of‐origin, 104. NoThere are a growing number of features and additional analytes including EVs, ctRNA, proteins and other components that are being explored in various cancer types that are beyond the scope of this current review. Many of these have yet to be thoroughly evaluated in lymphomas, although in time they may demonstrate tremendous added potential in combination with ctDNA genotyping.7Our understanding of ctDNA and its dynamics during the lymphoma disease course from diagnoses through treatment to progression has rapidly accelerated in a relatively short period of time and continues to expand. Published studies on the potential utility of ctDNA assessment in lymphomas have been in relatively small patient cohorts and there is a need for more detailed evaluations in much larger contemporary patient cohorts across the breadth of lymphoma subtypes. There remains a need for rigorous standardisation, harmonisation, and quality assurance processes together with the development of adequately sensitive assays especially in the setting of MRD. The current wave of clinical trials with integrated ctDNA profiling in lymphoma will be invaluable in demonstrating the feasibility, cost effectiveness, and utility in eventually bringing liquid biopsy analyses into our routine clinical workflows.Edward Poynton and Jessica Okosun reviewed the literature, wrote the manuscript and approved the final version.The authors declare that they have no conflict of interest.


Population diversity is a reality in our societies and requires health systems and health professionals to adapt to the needs of diverse patient groups, including migrants and ethnic minorities. This study aims to investigate topics and methods that should be prioritised in an online course on diversity competence in healthcare delivery to improve health care encounters and provide health services that meet the unique needs of all patients in order to reduce health disparities.The study uses an adapted Delphi method including two rounds, combining some open-ended questions with pre-defined items, asking 31 European academic experts and health professionals within the field of migrant health to rate training content and teaching methods. Consensus for training topics was set to 80% and for teaching methods 70%.The only item reaching 100% consensus as being important or very important to include was ‘health effects of migration ’. Other high-scoring items were ‘social determinants of health’ (97%) and ‘discrimination within the healthcare sector’ . A general trend was to focus more on reflective practice since almost all items in the reflection section reached consensus. ‘Reflection on own stereotypes and prejudices’ reached the highest consensus in this section (97%).Experts’ prioritisations of teaching content and methods for diversity training can help the design of short online trainings for health professionals and reduce extensive course content, thereby fostering professional development and enabling diversity competence trainings to be implemented in cases of scarce resources.• Trend toward more focus on ‘diversity’ and less focus on ‘culture, and the inclusion of social determinants of health and awareness of stereotypes and bias in training of health professionals.• Diversity competence training should use reflective exercises and activities as teaching methods in online training.


Advances in paediatric hepatology have led to the increasing survival of patients with paediatric‐onset chronic hepatobiliary disease into adulthood. Data are lacking with regard to the outcomes of this heterogeneous group of patients and current transition models may be insufficient. This retrospective regional cohort study examined the outcomes of these patients cared for in a paediatric gastroenterology centre following transfer to adult services.A prospective database of paediatric patients with liver disease identified those already transferred to adult services. Following exclusions, medical notes were examined and health parameters recorded including initial diagnoses, transplant status, fertility and mortality. Descriptive statistics were used to calculate follow‐up data and transplant‐free survival (TFS).Overall, 63 patients  entered the final analyses with a median follow‐up of 27.5 years. The most common diagnosis was biliary atresia (19%); 27 different diagnoses were apparent within the cohort highlighting the heterogeneity within a single centre. Transplant prevalence at adult transfer was 41%; 14% of patients were lost to follow‐up including 10% of transplant patients. TFS for biliary atresia was 17% after 37.4 years follow‐up and was 54% for the total cohort. There were seven documented pregnancies and the prevalence of any psychological or psychiatric input was 44%. Transplant complications occurred in 38% of patients; there were two cancer diagnoses and two deaths following transfer.Although overall mortality was low, the health‐care burden of patients with paediatric‐onset chronic liver disease is high. This group is also very heterogeneous, making structured transition to adult services difficult. The long‐term outcomes of children following liver transplantation and with specific conditions are largely known.The heterogeneity and outcomes of population‐based cohorts of children with chronic liver disease transitioning to adult centres from their local gastroenterology unit are unknown.Children with chronic liver diseases have significant morbidity in early adulthood and their overall health‐care burden remains high.Children with chronic liver diseases represent a very heterogeneous group and may require condition‐specific transition models.Advances in paediatric hepatology throughout the 1980s and 1990s have led to the increasing survival of patients with paediatric‐onset chronic hepatobiliary disease into adulthood.more representative of the majority of centres transitioning this patient group, without the risk of transplant centre bias.This retrospective regional cohort study aimed to assess the basic health outcomes of patients with paediatric‐onset chronic liver disease from a tertiary paediatric gastroenterology centre following their move to adult services. Given the lack of published guidance during the study period, a focus on the transition process itself was deliberately avoided. Overall, consisting of a relatively small and heterogeneous group of patients, this cohort was felt to in fact be Within Scotland (and throughout the United Kingdom (UK)), all tertiary paediatric gastroenterology units are loosely affiliated with one of the three quaternary paediatric liver transplant centres . Centralisation of services occurred in the early 2000s and although a specialist paediatric hepatologist will often attend satellite clinics within these tertiary centres to help monitor these patients: (i) the day‐to‐day care of these patients remains under the direct guidance of the tertiary gastroenterologist; (ii) some patients may never be known to, or have a face‐to‐face meeting with, the quaternary specialist; and (iii) the majority of adolescent patients will transition to the care of an adult gastroenterologist/hepatologist locally with no input from quaternary centres.ad hoc with the local adult hepatology unit from 1997 to 2001 with more straightforward patients transferred by way of a single transfer letter. Since 2002, a more formal transition process has been adopted with a single clinic, attended by a paediatric gastroenterologist, adult hepatologist and specialist nurses, held in RHSCE each spring followed by an identically structured MDT clinic held in the local adult hepatology centre in the autumn. Due to the time frame of this study, the process of transition to adult services has changed as new evidence became available. In the UK, patients would often remain in paediatrics until they leave secondary education (around 17–18 years of age), but a proportion of patients transition earlier due to several factors. Throughout the study period, assessment readiness for transition using a formal tool was not used; however, nurse specialists would meet the young person separately around the time of transition to discuss drug compliance, understanding of medications and their side effects, the disease itself and other issues relating to sexuality, education, smoking and alcohol. In a small number of cases, a single transfer letter still remains the only feasible mechanism of paediatric to adult transfer.The Department of Paediatric Gastroenterology and Nutrition at the Royal Hospital for Sick Children, Edinburgh  was established in August 1997. The hospital has always acted as a referral centre for all hospitals in South‐East Scotland (SES). RHSCE is not, and never has been, a paediatric liver transplant centre. With regard to adult transition, this was performed Since 1st August 1997, RHSCE has been prospectively collecting data on paediatric patients with chronic liver disease and now holds details of over 350 patients. From this database, patients 16 years and over by 31st December 2018 (hereafter referred to as ‘study end’) were identified and excluded if they: (i) had moved out of the region while still in paediatric care; (ii) died while in paediatric services; (iii) were discharged at or before transition/transfer to adult services; (iv) were still under the care of paediatric services at study end; or (v) were living outside of the SES region as described above based on postcode at study end. Accurate incidence and prevalence data for all paediatric chronic liver diseases are not available either in the UK or elsewhere.Clinical data were collected from electronically stored letters and the TrakCare Electronic Medical Record System ; data were then collated in Microsoft Excel 2007 . Due to the historical nature of some medical records, the precise date of diagnosis was not always available. In these cases, the following criteria were used as the date of diagnosis: (i) first mention of the confirmed paediatric liver disease diagnosis in correspondence; (ii) the date of Kasai procedure for patients with biliary atresia; (iii) for hepatitis B and C patients their oldest correspondence with mention of their diagnosis.  Transfer was defined as patients who had moved directly from paediatric to adult services, with a single transfer letter, with the date of transfer being the last paediatric clinic appointment. Transition was defined as the formal process described above with the transition date taken as the first joint paediatric and adult transition clinic appointment. Patients were classed as lost to follow‐up if there were at least three non‐attendances to clinic and/or no tertiary health‐care interaction within the past 3 years by study end. Patients discharged to the care of their community general practitioner while in paediatric services and patients under the care of the department of Inherited and Metabolic Diseases (who transition patients independently) were excluded. All patients (if compliant with follow‐up arrangements) have regular bloods  and imaging as part of their routine follow‐up, especially with regard to hepatocellular carcinoma screening.Descriptive statistics of the cohort were calculated using Microsoft Excel . Median and interquartile range (IQR) were used for non‐parametric variables. Transplant‐free survival (TFS) rates were calculated at the point of adult transfer and also calculated at the point of last known follow‐up or study end, whichever was latest. TFS in adult services ended at liver transplantation, study end or death, whichever was latest.Clarification with the South‐East Scotland Ethics Scientific Officer established that formal ethical approval was not required for this type of retrospective study. However, a Children's Services' project proposal and registration form were approved by the RHSCE Quality Improvement Team. This study conforms to the provisions of the Declaration of Helsinki.Initial screening identified 101 patients to be 16 years and over at study end; 63 patients entered the final analyses . The six patients with portal hypertension had the following diagnoses: cavernous transformation of the portal vein (n = 2); idiopathic portal vein thrombosis (n = 2) and hepatoportal sclerosis secondary to thiopurines following treatment for acute lymphoblastic leukaemia (n = 2). There was one patient with progressive familial intra‐hepatic cholestasis type 1  and one patient with BSEP deficiency (confirmed mutation in ABCB11). Further highlighting the rarity of most chronic paediatric liver diseases, the miscellaneous group consisted of 11 patients with unique diagnoses within the cohort: autoimmune sclerosing cholangitis, blunt liver trauma, choledochal cyst, congenital biliary hypoplasia, focal nodular hyperplasia with a congenital portocaval shunt, hepatitis B, hepatoblastoma, intestinal failure‐associated liver disease, Jeune syndrome, non‐alcoholic fatty liver disease (NAFLD) and unilateral naevoid telangiectasia.Final diagnoses of the cohort are shown in Figure While in paediatric services, there were a total of 29 transplants performed in 26 patients Fig. . Median Overall, 14 patients were transferred and 49 underwent formal transition at a median age of 17.2 years , biliary atresia (n = 2), alpha‐1‐antitrypsin deficiency (n = 3) and idiopathic portal hypertension (n = 1). Importantly, among these, three of the patients had undergone liver transplant at transition ; therefore, overall 10% of transplant patients were lost to follow‐up in tertiary services. However, these patients may have remained under the care of their community general practitioner.The median age of the entire cohort was 26.3 years  at study end. Following transition/transfer to adult services, seven patients moved out of the region and nine were lost to follow‐up. Notably, no patient was formally discharged from follow‐up for their liver disease. The combined follow‐up patient‐years from diagnosis was 1753 patient‐years with median follow‐up per patient of 27.5 years (IQR 19.5–37.3). For those lost to follow‐up, median time in adult services was 4.5 years (IQR 2.9–11.1). The diagnoses in those patients who were lost to follow‐up were hepatitis C (The TFS for the cohort at study end was 54% (34 of 63). The TFS for patients with biliary atresia was 17% (2 of 12) at a median of 37.4 years (IQR 33.2–42.0) from diagnosis. There were six patients with nine documented pregnancies  during follow‐up. A summary of these patients and their pregnancies are shown in Table Within the cohort, two patients underwent re‐transplant in adult services; one patient with an initial diagnosis of fulminant liver failure of unknown cause developed liver cirrhosis and hepatocellular carcinoma and a patient with Alagille syndrome developed acute‐on‐chronic rejection while on mycophenolate mofetil. Of the three patients that had their first liver transplant in adult services, their diagnoses were biliary atresia, congenital biliary hypoplasia and alpha‐1‐antitrypsin deficiency Fig. . Their fKlebsiella pneumonia; both patients died in their 30s. Overall mortality in the cohort was 3%.There were two deaths within the cohort: (i) a patient with Crigler–Najjar syndrome who developed post‐transplant lymphoproliferative disorder; and (ii) a patient with congenital hepatic fibrosis who was subsequently reclassified as Caroli syndrome in adult services (non‐transplanted) and who suffered from a cerebral haemorrhage on a background of chronic renal failure and who developed acute respiratory failure secondary to In this heterogeneous, but representative, population‐based regional cohort study of patients with paediatric‐onset liver disease, we have clearly demonstrated the high health‐care utilisation that these patients face in early adulthood. Specifically, we have shown the diverse number of individual diagnoses  but the significant prevalence of transplantation at transfer (41%). Our data on TFS (54%) are novel for this heterogeneous group of patients and is in‐line with recent data with regard to biliary atresia.The strengths of this study are the prospective nature of the original cohort, the long follow‐up duration (median > 27 years), the strict geographical nature of the cohort  and the broad overview of health‐related outcomes. A broad and non‐systematic review of the current literature on PubMed (using the search terms: ((‘Follow‐Up Studies’[Mesh]) OR ‘Survival Analysis’[Mesh]) AND (‘Liver Diseases’[Mesh]) AND ) produced no regional cohort studies published in the last decade outlining the natural history of this heterogeneous group of patients. Although there are a significant number of studies evaluating single conditions  and a detailed analysis of liver function in certain scenarios (such as pregnancy) was not available. We however feel that individual alterations in liver function are multifactorial and this would not have been a useful analysis to perform. Data on medication adherence was also not available as this is not possible (or indeed accurate) in this retrospective design. Although covering a quarter of the Scottish population, the relative rarity of paediatric‐onset chronic liver disease also means this patient cohort remained relatively small and conducting similar studies in other regional centres may be useful. It is also of interest that, again likely due to the time frame of the study, only one patient had a diagnosis of NAFLD. Given that there was almost no published research in the field of paediatric NAFLD prior to 2008, and with these patients possibly looked after in general paediatric or endocrinology clinics in the past, it is likely that future data will comprise a much larger proportion of these patients.Health‐care professionals in adult services have the potential to encounter treatment‐related complications soon after transition compared to chronic liver diseases of adult‐onset.Structured transition to adult services is now being seen as a crucial process and normal standard of care in patients with chronic disease,As patients move from a more paternalistic and interdisciplinary approach to a more independent and autonomous practice in adult care,Using a population‐based cohort study design, we have demonstrated the heterogeneous nature of paediatric‐onset chronic liver disease and health‐care issues encountered following handover to adult services. Further population‐based longitudinal cohort studies are required to further evaluate this group of patients to ensure that a more personalised transition programme can be adopted to improve long‐term follow‐up and reduce morbidity and mortality.


Flower phenotypes evolve to attract pollinators and to ensure efficient pollen transfer to and from the bodies of pollinators or, in self‐compatible bisexual flowers, between anthers and stigmas. If functionally interacting traits are genetically correlated, response to selection may be subject to genetic constraints. Genetic constraints can be assessed by quantifying standing genetic variation in (multivariate) phenotypic traits and by asking how much the available variation is reduced under specific assumptions about phenotypic selection on functionally interacting and genetically correlated traits.Dalechampia blossoms. First, we used data from a greenhouse crossing design to estimate the G matrix for traits representing the relative positions of male and female sexual organs (anthers and stigmas) and used the G matrix to ask how genetic variation is distributed in multivariate space. To assess the evolutionary importance of genetic constraints, we related standing genetic variation across phenotypic space to evolutionary divergence of population and species in the same phenotypic directions.We evaluated multivariate evolvability and potential genetic constraints underlying the evolution of the three‐dimensional structure of Evolvabilities varied substantially across phenotype space, suggesting that certain traits or trait combinations may be subject to strong genetic constraint. Traits involved functionally in flower‐pollinator fit and autonomous selfing exhibited considerable independent evolutionary potential, but population and species divergence tended to occur in phenotypic directions associated with greater‐than‐average evolvability.These results are consistent with the hypothesis that genetic constraints can hamper joint trait evolution towards optimum flower‐pollinator fit and optimum self‐pollination rates. Evolution of complex phenotypes is commonly governed by multiple conflicting selection pressures interacting with genetic constraints . Studies of G matrices for complex phenotypes have revealed that genetic variance is often unevenly distributed across phenotype space, so that some phenotypic axes (trait combinations) are associated with above‐average evolvability, while others exhibit little or no evolvability . We then used the G matrix and the multivariate evolvability framework of Hansen and Houle  has been a long‐term model system for investigating pollinator‐mediated floral evolution. The woody vine D. scandens L. s.l. is typical of the genus in having unisexual male and female flowers aggregated into functionally bisexual blossom inflorescences  for gland–stigma distance, gland–anther distance, and anther–stigma distance, three key traits that jointly describe the relative positions of male and female sexual organs , plants (j), dates (k), and individual blossoms (l). To estimate the G matrix (G), the “animal”‐level random effect was distributed as a~N, where A is the additive relatedness matrix derived from the breeding design, and ⊗ is the Kronecker product , measurement dates (d), and blossoms within plants (q). We sampled the posterior distribution for 750,000 iterations with a transient of 250,000 iterations discarded and a thinning interval of 500 and confirmed convergence by evaluating posterior trace plots and confirming effective sample sizes close to the expected 1000.To estimate the β is defined as the predicted evolutionary response to selection in the direction of β. To quantify multivariate constraints arising from the quantitative‐genetic architecture of complex phenotypes as represented by the genetic variance matrix G, Hansen et al. (β when other defined phenotypic traits are held constant (as when these are under stabilizing selection). In the present study, quantifying conditional evolvabilities allowed us to explicitly evaluate, for example, how much the realized evolvability of anther–stigma distance is reduced assuming stabilizing selection on the flower‐pollinator fit traits . The proportion of genetic variance available while conditioning on the constraining traits is referred to as autonomy.Hansen and colleagues  of the eigenvalues, and the mean of the univariate evolvabilities .We used functions from the evolvability R package , population divergence, and species divergence following the approach of Bolstad et al. , populations (j), plants (k) and individual blossoms (l). The population‐level random effect estimates the variance matrix DP for population‐mean phenotypes across populations. The phenotypic measurements were ln‐transformed before analysis, and the estimated variances and covariances are therefore on a proportional scale comparable to the within‐population evolvabilities.To assess the role of genetic constraint in the evolution of floral architecture in d et al. . At the DS, we fit a similar model to field measurements from 43 populations of 11 Dalechampia species , and the D matrices describe proportional variance among populations or species. The genetic‐constraints hypothesis predicts a positive relationship between proportional evolutionary divergence and evolvability  of 2.59% can be interpreted as the expected proportional response to an episode of unit‐strength selection and translates into an evolutionary doubling‐time of t2 = ln(2)/0.0259 = 26.8 generations under unit strength selection.Univariate evolvabilities differed markedly among the three original traits Table , and theThe moderately strong genetic correlations between the three focal traits translated into a median autonomy of 53.3% Table , which mExploring patterns of evolvability across randomly chosen directions in phenotypic space Figure  revealedComparing the extent of population divergence among the original traits and among directions in phenotypic space revealed an overall positive relationship, and the most evolvable focal trait (anther–stigma distance) has diverged the most among populations Figure . DirectiWhen focusing on the divergence of species and thus extending the time‐scale and extent of divergence, the positive relationships between divergence and evolvability remained. However, at the species level, both pollinator‐fit traits have diverged to approximately equal extents, despite greater evolvability of gland–stigma distance than gland–anther distance Figure .Dalechampia blossom traits involved in flower‐pollinator fit and autonomous selfing exhibit appreciable independent evolutionary potential, but also that populations and species have diverged to a greater extent in those phenotypic directions associated with greater multivariate evolvability. The latter result is consistent with the hypothesis that joint trait evolution toward optimum flower–pollinator fit and optimum self‐pollination rate is subject to genetic constraints, thus possibly providing a partial explanation for the observed distribution of trait values across the genus . This result confirms the unusually high evolvability of herkogamy  is consistent with a previous literature survey . One way forward would thus be to combine estimates of population divergence, (multiple) G matrices, and multiple selection estimates  of Dalechampia taxa included in the analysis of species divergence.Click here for additional data file.


Sustainability of Australian healthcare is not assured. Health costs as a fraction of GDP continue to outstrip inflation, as demands spike from an ageing population and prolonged health crises stretch budgets.appropriate workforce is present in an appropriate geographic distribution for equitable health care access.Non‐capital resources in surgery involves ensuring the needs of the workforce to provide surgical care to the community are met. Workforce planning has failed to co‐ordinate the complete pipeline from training medical graduates to employment of surgeons in the public sector. This has created a challenge to ensure an The surgical workforce is modelled by governments to meet future demands. However, there is no systematic co‐ordination between the university sector that trains medical graduates, federal and state governments or RACS to plan public funding to Specialist Training Positions (STP) to match the number of medical graduates interested in surgical training. A lack of uniformity of points criteria for approved courses for Surgical education and training (SET) application set by different specialty training boards reduce transferability between specialties for junior trainees, prolongs entry and disincentivise diversity of prior experience in the applicants for surgical training which may not ultimately reflect the necessary scope of practice required to service the community. Consequently, a boost in medical graduate training without proportionate funding for surgical training or services has created unprecedented pressures on applicants for surgical training who take longer to enter training, face increased career uncertainty and are vulnerable to exploitation.Reduced access for younger fellows in the public sector incentivises additional post‐fellowship training to qualify for highly specialized but limited metropolitan positions. Thus, fellows who complete training at an older age, with more academic qualifications, larger debt, less mobile families and reduced job opportunities in the public sector are incentivized to work in geographical regions with improved socioeconomic status, potentially increasing health care access inequity.Investment in training represents a large non‐capital cost. However, reducing attrition and burnout ensures return on investment with estimated savings in the US of USD $6.3 billion.Paralleling untenable pressures on human resources is a critical unsurpassed demand on limited capital resources. As costs of new interventions rise and surgical budgets strain, health systems can respond by (i) reducing unnecessary cost by implementing value‐based healthcare models,Crisis management increases demand on capital and non‐capital resources and exacerbates health inequity. As seen during COVID‐19, crises necessitate prioritization of acute health care, which has a disproportionate impact on non‐urgent surgery. If the system is overwhelmed with numerous crises prolonging the impact of the shock to routine care, overcoming subsequent backlogs places unsustainable pressure on the workforce which impacts rural areas and areas with poor socioeconomic status due to limited workforce availability and high demand on public health services. Public‐private partnerships globally have been utilized as a short‐term strategy to overcome backlogs but they divert money away from development of local infrastructure and workforce expertise needed to deliver longer‐term sustainable public care.Environmental resources, not typically considered as financial costs, are hidden areas of unsustainable cost. Climate emergencies such as the bushfires of 2019 have demonstrated the magnitude of this on workforce and health care burden, estimated in the order of hundreds of billions of dollars.Increasing concern about the sustainability of Australian surgical care necessitates measures to reduce waste as an economic and ethical priority. COVID‐19 has provided a unique opportunity to press the reset button, examine areas of weakness and implement change (Table Gareth Weijie Crouch: Conceptualization; methodology; writing – original draft; writing – review and editing. Payal Mukherjee: Conceptualization; methodology; writing – original draft; writing – review and editing. Arthur Richardson: Conceptualization; supervision; writing – review and editing.


Research is a cornerstone of evidence‐based dietetic practice. Research skills are often taught at university through experiential learning during a final‐year research project. The present study aimed to investigate attitudes towards research and confidence in research skills among student dietitians before and after a research project.A questionnaire survey of student dietitians’ attitudes to research and confidence in research skills was undertaken before and after completing a research project at two universities in London, UK. Dichotomous data were compared before and after the research project using a McNemar's test. Factors associated with ‘high confidence’ or ‘improved confidence’ in overall research skills at the end of the research project were investigated using multivariable logistic regression.p < 0.001), and 113 (70.6%) reported ‘improved confidence’ in overall research skills. The only factor associated with ‘high confidence’ in overall research skills was having ‘high levels of involvement in the overall research process’ .In total, 160 student dietitians completed a questionnaire before and after their research project. The majority had positive attitudes to research both before and after their research project. There was an increase in numbers with ‘high confidence’ in overall research skills before  and after  the research project (Student dietitians have positive attitudes towards research and undertaking a research project significantly improves confidence in their research skills. A higher level of involvement in the research project is the single most significant factor associated with high confidence in research skills.  This is the largest study to date of student dietitians’ experiences of undertaking a research project during their university degree.Student dietitians have positive attitudes to research both before and after their research project.Research projects improve student dietitians’ confidence in their research skills.The single most significant factor associated with developing high confidence in research skills is having a greater level of involvement in the project. Research is a cornerstone of evidence‐based dietetic practice. Research is essential to provide the evidence for diet–disease relationships, for the effectiveness of interventions and to provide evidence of the impact of dietitians.Professional associations or governing bodies have standards for dietitians that include competency in research and evidence‐based practice in Australia,In the wider healthcare professions, generally positive attitudes towards involvement in research have been reported.Barriers to research involvement in dietetics have been extensively examined and consistently report a lack of time and lack of funding as key issues, but negative attitudes towards research and lack of confidence in research skills have also been identified as important obstacles to research involvement.The reported lack of sufficient research training in pre‐registration dietetics programmes coupled with evidence that education, knowledge and interest in research are associated with future research involvement among dietitiansIn the UK, pre‐registration programmes in dietetics require student dietitians to have a ‘critical and applied knowledge and understanding of research …’ including, amongst others: research ethics and research governance; the principles of scientific enquiry; quantitative and qualitative research design; data management; statistical analysis and interpretation; critical appraisal; and evidence informed practice.A small number of studies have demonstrated that student dietitians’ exposure to undertaking a research project improves confidence in their research skills.The present study aimed to investigate attitudes towards research and confidence in research skills among student dietitians before and after a research project. Specifically, the study investigates: (i) attitudes towards research and confidence in research skills among student dietitians; (ii) the effect of undertaking a final year research project on these attitudes and confidence; and (iii) the factors associated with ‘high confidence’ and ‘improved confidence’ in overall research skills.The study used a questionnaire to investigate the confidence and attitudes of student dietitians before and after undertaking their final‐year research project.Data were collected from students before and after completing a compulsory research project module in their final year of the 4‐year BSc Nutrition and Dietetics programmes at King's College London and London Metropolitan University, two large universities in London, UK. All students undertaking the research project module were eligible, with no exclusion criteria, and all were approached over five consecutive years at King's College London (academic years ending in 2005–2009) and three consecutive years at London Metropolitan University (academic years ending in 2005–2007), aiming to recruit a sufficiently large, unselected and representative sample.Both universities provide students with a compulsory ‘research methods’ module earlier in the BSc degree (Years 1 or 2) in preparation for the research project module in the final (Year 4). The research project modules involve some research methods teaching  followed by undertaking a research project part‐time and under supervision for 3–4 months.Project selection consisted of compilation of a list of research projects proposed by academic staff at the university or by an external dietitian or nutritionist who would co‐supervise a project with an internal academic.Following project allocation, all research projects were conducted according to standard university procedures. A research team was formed consisting of the student dietitian and one (or more) university academic and, for external collaborative projects, the external dietitian/nutritionist. Each research team had a preliminary meeting to discuss the project, team member responsibilities, supervisory arrangements and the dissemination of results.On completion of the research projects, students wrote a dissertation and prepared and presented a research presentation or poster or participated in a viva voce in line with the assessment requirements of their university.Students were invited to participate in a survey of their attitudes and confidence in their research skills by completing a questionnaire before (first lecture of the module) and after undertaking their final‐year research project (following submission of their dissertation and completion of their assessment). The questionnaire was developed by the three members of the research team  with extensive experience of questionnaire design, psychometric testing and survey conduct. Questionnaire design consisted of performing a literature search to identify previously conducted surveys, where possible, based upon which a bespoke questionnaire was developed. The first iteration of the questionnaire was piloted in 36 students and amendments were made following feedback and discussion, and the data collected from the pilot questionnaire were not included in the current analysis.Attitudes towards research were measured using a series of nine statements reflecting a range of positive and negative attitudes towards research and were taken directly from a previous questionnaire, which, at the time of the study design, was one of the few studies of dietitians’ attitudes towards research.Confidence in research skills was measured for 10 research activities based upon the different stages of research process from a model provided by the National Institute of Health Research at the time . Minor adaptations were made; namely, removing a question on obtaining research funding : changing ‘Report on the study and disseminate the findings’ into two processes ‘Writing a research report’ and ‘Orally presenting research findings’ (as both were performed as part of the research project modules and require distinct sets of skills); and, finally, a question regarding confidence in the ‘Overall research process’ was added . Confidence was rated on a five‐point Likert scale . As well as presenting data on confidence in each of the 10 activities and in the overall research process, data were also collapsed to report those with ‘high confidence’ . The numbers of students with ‘improved confidence’ were calculated, defined as increasing in confidence by one or more on the Likert scale between baseline and after completing the project .The questionnaire completed at the end of the research project also examined the extent to which students were involved in different aspects of research during their project and their perceptions of whether the project had contributed to the development of their research skills. These two outcomes were measured for the 10 research activities and the overall research process and were rated using the same five‐point Likert scale . High levels of involvement were defined as the student reporting ‘4, A lot of involvement’ or ‘5, A great deal of involvement’ in the research project.The study received ethical approval from the research ethics committees of both King's College London and London Metropolitan University. Questionnaires were distributed during lectures in sealed envelopes and completed on a voluntary basis. Questionnaires were anonymised using a code such that questionnaires before and after the research project could be paired for analysis but did not contain the students’ name or any identifiable details. Questionnaire responses were not linked in any way to student records or academic results and did not contribute or impact in anyway on the assessment of the research project module.n (%). Dichotomised data were compared between baseline and after the research project using a McNemar's test.Data were only analysed for students who provided a questionnaire at both baseline and following completion of the project, in order that changes in attitudes and confidence could be calculated for all respondents. Descriptive statistics were calculated for the variables of interest and data are presented as p < 0.1 in the univariate logistic regression model together with all project‐related characteristics were included in the multivariable logistic regression model.The two major educational outcomes of interest were having ‘high confidence’ in overall research skills after the research project  and ‘improved confidence’ in overall research skills between baseline and after the research project . These two educational outcomes were explored for related factors using univariable logistic regression. Eight variables were entered into the model relating to personal characteristics  and project‐related characteristics . Personal factors with a p < 0.05 was considered statistically significant.Data were analysed in SPSS, version 26 (IBM Corp.). Questionnaires were distributed to 196 students, of whom 184 (93.9%) returned a questionnaire at baseline and 166 (84.7%) returned one following completion of the research project. In total, 160 (81.6%) students returned a questionnaire at both time points and are the complete case analysis study population reported here.The 160 students had a median age of 25.0 years (interquartile range = 10.7) and 149 (93.1%) were female (Table p < 0.001). The numbers whose attitudes improved after the research project ranged from 10.6% (‘I find it hard to interpret research’) to 26.3% (‘I don't see research as part of a dietitian's job’). In terms of attitudes towards future involvement in research, after the research project, a very small minority of students (5.6%) said they ‘did not want to be involved in audit and research’ and the majority (63.1%) said they wanted ‘to take part in research with colleagues’ (Table The majority of student dietitians’ responses to statements reflected positive attitudes towards research at baseline and after completing the project Table . The onlp = 0.01) and ‘hope to do research with colleagues’   in their research skills was very low at baseline, ranging from only 6.9%  to 35.6%  . The exception was ‘obtaining research ethics approval’ (28.8%).The areas of the research project in which the most students had high levels of involvement  were ‘completing a literature search’ (91.3%), ‘writing a research report’ (95.6%) and ‘drawing conclusions from research’ (95.0%). The areas where fewest students had high levels of involvement were ‘obtaining research ethics approval’ (11.3%), ‘designing a research protocol’ (42.5%) and ‘deciding on data collection methods’ (38.8%). In total, 133 (83.1%) students reported high levels of involvement in the overall research process (Table Over three‐quarters of students (77.8%) reported that undertaking their research project had contributed ‘a lot’ or ‘a great deal’ to the development of their overall research skills Table . The majp = 0.035) and the student reporting ‘high levels of involvement in the overall research process’ . However, following multivariable logistic regression the only factor significantly associated with high confidence in overall research skills was having ‘high levels of involvement in the overall research process’ . The multivariable regression model was statistically significant .In total, 79 (49.4%) students had high confidence in their overall research skills after the research project, and the odds of this outcome was analysed using logistic regression Table . Followip = 0.027) and ‘undertaking original data collection’ . However, following multivariable logistic regression, no factors were statistically significant associated with having improved confidence in research skills and the model itself was not statistically significant .In total, 113 70.6%) students reported improvement in their overall research skills after the project, and the odds of this outcome was also analysed using logistic regression , the type of project  and whether original data was collected, many of which are themselves related, highlighting the importance of multivariable regression analysis to test for independent associations. Indeed, the only factor independently associated with high confidence in the overall research process was having high levels of involvement in the overall research process. This has been previously alluded to in the wider literature outside of health professional education. For example, a previous study shows greater perceived benefit to skills when undertaking research projects of greater duration,It is encouraging that the only factor related to high confidence in overall research skills was high levels of involvement in the overall research process, which resulted in a more than six‐fold greater odds of high confidence and was more important than other project‐related factors . Offering variety in opportunities in project type can be challenging in some university settings as a result of access to laboratory facilities, the research interests of academic staff and the existence of established external collaborations. However, the present study reinforces that, irrespective of these other factors, the most important goal is to offer research projects that enable as much involvement in the overall research process as possible, thus emphasising the role of experiential learning (learning by doing) in research skills development.Reassuringly, personal characteristics  were not significantly associated with high confidence or improved confidence. Two project‐related factors are worthy of comment: the supervision model/location and the project type. Projects that were undertaken externally and supervised by both an internal and an external supervisor were associated with greater odds of high confidence on univariable but not multivariable analysis. The positive effect of collaborative supervision is congruent with previous studies.Research projects vary widely in the extent to which they involve different activities in the research process. Given the relationship between high levels of involvement and high confidence in research skills, the limited confidence in specific skills such as ‘developing a hypothesis’ and ‘obtaining research ethics approval’ is unsurprising. The short duration of the research project  means that students are seldom involved in the early stages of project conception, design and approval. As such, these areas where involvement and confidence are low, may benefit from being further incorporated into the teaching curriculum and into future research projects. In addition, on circulating research project titles, supervisors could indicate the likely levels of involvement in different aspects of the research process so that students can select a project more likely to fit their learning needs, as well as their interests.Undertaking a final year research project has been described as a ‘high impact educational experience’In the present study, all students undertook a research project; however, it would be interesting to compare the attitudes and confidence in research skills of those who do (intervention) and do not (control) undertake a research project. Such a control group would be challenging in the UK environment where a research project is commonly a compulsory component in the final year of a Bachelors degree in dietetics. In other countries, including Australia, Canada and the USA, such comparisons can be made; however, students are not randomly selected to do a research project or not, and so selection bias might result in the most research‐orientated students carrying out a research project and therefore would impact conclusions.This is the largest study directly comparing student dietitians’ attitudes to research and confidence in their research skills before and after undertaking a research project. It has used identical questionnaires items and response sets at baseline and after the project to enable direct comparison. However, the questionnaires were not validated prior to use, although the attitude statements were adapted from a previous survey investigating dietitians’ attitudes towards research.The large sample size enabled investigation of the factors independently associated with high confidence and improved confidence through multivariable logistic regression. The lack of exclusion criteria and high response rate (81.6%) minimised selection bias. Using a complete case analysis of only those students who completed a questionnaire at both time points avoided the need for data imputation, although this may have resulted in the inclusion of only those students who successfully completed the research project module, and therefore may have inflated improvements in confidence in research skills. The sample was mostly young and mostly female, although this is largely representative of student dietitians in the UK. Although the questionnaire surveys were conducted contemporaneously to the research projects, these were conducted more than 10 years ago. However, we do not consider this will impact data integrity and interpretation because the research project modules are still undertaken in the same style at the two universities and there is no reason to assume that student cohorts have changed during this period. Furthermore, this evaluation was performed at only two universities and, although there may be differences in the content and delivery of research training provided compared to other universities, the quality of the research training in pre‐registration dietetics programmes is likely representative of other universities in the UK.The present study is also limited in that it only measured students’ self‐reported confidence in research skills. Student reports of research skills has been shown to lack agreement with academic and performance‐based assessment of research skills.Finally, the data collected were exclusively quantitative. Qualitative enquiries may help bridge the gap between the generally positive attitudes toward research and actual research involvement, as well as provide in‐depth insight into how the research project may impact attitudes to research and confidence in research skills.Student dietitians exhibit generally positive attitudes towards research. In general, undertaking a research project did not improve attitudes to research but did improve students’ confidence in their research skills. High levels of involvement in the overall research process was the only significant factor associated with high confidence in overall research skills. This suggests that, regardless of personal or project‐related characteristics, so long as student dietitians are highly involved in the research process, they have six‐fold greater odds of achieving high confidence after undertaking a research project. Whether these improvements in confidence are sustained and translate into future research involvement in practice warrants further investigation.Conceptualisation, study design, and data collection: Kevin Whelan, Jane E. Thomas and Angela M. Madden. Data analysis and interpretation: Kevin Whelan, Kate R. Castelli, Camilla Trizio and Oliver Howard. Manuscript preparation: Kevin Whelan. Manuscript review and approval: Kevin Whelan, Kate R. Castelli, Camilla Trizio, Oliver Howard, Jane E. Thomas and Angela M. Madden. All authors critically reviewed the manuscript and approved the final version submitted for publication.The authors declare no conflicts of interest.The authors affirm that this manuscript is an honest, accurate and transparent account of the study being reported. The authors affirm that no important aspects of the study have been omitted and that any discrepancies from the study as planned have been explained.The study received ethical approval from the research ethics committees of both King's College London and London Metropolitan University.


Acrocephalus arundinaceus) using pedigree‐based linkage mapping. This species has extended Z and W chromosomes (“neo‐sex chromosomes”) formed by a fusion between a part of chromosome 4A and the ancestral sex chromosomes, which provides a unique opportunity to assess recombination and sequence evolution in sex‐linked regions of different ages. We assembled an 87.54 Mbp and 90.19 cM large Z with a small pseudoautosomal region (0.89 Mbp) at one end and the fused Chr4A‐part at the other end of the chromosome. A prominent feature in our data was an extreme variation in male recombination rate along Z with high values at both chromosome ends, but an apparent lack of recombination over a substantial central section, covering 78% of the chromosome. The nonrecombining region showed a drastic loss of genetic diversity and accumulation of repeats compared to the recombining parts. Thus, our data emphasize a key role of recombination in affecting local levels of polymorphism. Nonetheless, the evolutionary rate of genes (dN/dS) did not differ between high and low recombining regions, suggesting that the efficiency of selection on protein‐coding sequences can be maintained also at very low levels of recombination. Finally, the Chr4A‐derived part showed a similar recombination rate as the part of the ancestral Z that did recombine, but its sequence characteristics reflected both its previous autosomal, and current Z‐linked, recombination patterns.Recombination strongly impacts sequence evolution by affecting the extent of linkage and the efficiency of selection. Here, we study recombination over the Z chromosome in great reed warblers ( That recombination is critical for maintaining the fitness of large genomic regions, becomes evident when recombination is lacking , while recombination proceeds in the homogametic sex . This phenomenon is observed across a wide range of species including animals, plants and fungi  belongs to Sylvioidea and earlier studies have presented an incomplete linkage map for the species based on microsatellites and AFLP, including the Z chromosome , where females in the vast majority of species do not recombine for most parts of their sex chromosome pair and consequently exhibit a small PAR. There are, however, exceptions, such as in some species of Palaeognathae  and the remaining reads were realigned with GATK 3.8–0  restriction site‐associated DNA (RAD) library construction, sequencing (Illumina 100 bp paired‐end), trimming and demultiplexing was performed by BGI  with three different pipelines . We chose this LOD score since it resulted in defined LGs with synteny to single chromosomes in zebra finch genome assembly  was repeated twice. We ran ten marker orderings (starting from random order each time) in the module OrderMarkers2 and chose the order with the highest likelihood. We used the Kosambi mapping function, utilized grandparent genotypes to phase the data and set recombination to occur only in males (recombination2 = 0). The latter setting applies to most of the Z but is not optimal for the PAR region where both sexes recombine. To be certain that this setting did not interfere with the marker ordering and the following anchoring of the scaffolds (see below), we also ran the marker ordering and anchoring without the assumed PAR scaffold , we also ran the marker ordering step separately for the PAR (9 SNPs) to be able to use more suitable settings  for this region. However, the OrderMarkers2 was unable to find the best marker order for the PAR as all 10 runs had identical likelihood values, but different marker orders. Therefore, we cannot present exact recombination rates for both sexes in this region and use marker order according to the physical map order along the scaffold.Scaffold ordering and orientation was done in ALLMAPS  based on their anchored (liftover) positions in the ALLMAPS output . The PAR region was also divided into 200 kbp bins (N = 4), but since we did not get accurate map distances for this interval, the PAR is included only in the comparisons between the recombination regions (see below). Bins overlapping with scaffold gaps were excluded from the analyses.We extracted the chromosomal region between the first and last markers in the male‐specific Z linkage map and divided this region into nonoverlapping 200 kbp bins . The bin‐specific recombination estimate was then the genetic distance (cM) within these point estimates (Supporting Information π), linkage disequilibrium (r2 and D′), Tajima's D, Fay and Wu's H, proportion of exonic and repeat sequences, and GC content. These genome characteristics were calculated for each 200 kbp bin.We studied recombination in relation to several variables expected to be associated with it: genetic diversity  from the same population as the pedigree samples. For details regarding extraction, library preparation and trimming of raw reads, see Sigeman et al. . MappingAcrocephalus stentoreus, NCBI BioProject PRJNA578893, sample ID: SRR16079857, Sigeman et al., Fay and Wu's  quantifihttps://github.com/brentp/pyfasta/).Other genome variables  were calculated from the annotated reference genome , we defined the border between the PAR and the sex‐linked Z and divided the Z chromosome into different recombination regions: PAR (where both sexes recombine), male‐recombining (MREC) and nonrecombining . Then, we analysed the relationships between recombination and genome characteristics by comparing these three recombination regions, which should describe the effect of lacking recombination particularly. The relative amount of recombination between these regions is assumed to be: PAR > MREC > NONREC. Next, we studied the effect of the amount of recombination by correlations within the MREC region . The physical position effect on recombination was tested by correlation with the distance to chromosome end and by comparing chromosomal regions . All statistical tests were performed in R v. 1.3.1093  were adjusted for multiple testing with Holm's  for the great reed warbler Z‐linked and PAR protein‐coding genes. We aligned the great reed warbler Z sequences together with orthologous CDS sequences from zebra finch and chicken, which were downloaded from the Ensembl BioMart database  and collared flycatcher. Moreover, scaffold 98 showed a chimeric pattern indicative of a mis‐assembly. One part had markers assigned to LG‐Z and showed synteny to the fused part of chromosome 4A , while markers on the other part were left unassigned  and showed synteny to chromosomes 19, 6 and 1 in the zebra finch. After this manual curation, the scaffold was split into one Z‐linked part  and one autosomal part , of which only 98a was included in LG‐Z  included 676 markers located on ten scaffolds. Eight of them were identified as Z‐linked in ParentCall2 , while two scaffolds showed autosomal patterns regarding female/male read coverage and SNP heterozygosity (scaffolds 181 and 217), as would be expected for markers in the PAR. Scaffold 181 was excluded from LG‐Z after manual curation since it was assigned with only one marker  and showed synteny to chromosome 1A in zebra finch, great tit  and the order remained the same when the PAR scaffold (217) was excluded  had a total map length of 83.69 cM corresponding to a physical region of 86.48 Mbp Figure . DividinU = 1040.5, p = .66; Figure The MREC region is located in each end of the neo‐Z chromosome and can therefore be divided into an ancestral‐ and an added‐Z. The recombination rate was 5.41 cM/Mbp for the former and 3.90 cM/Mbp for the latter . However, when the rates were compared between these regions using the 200 kbp data, they were not statistically different . The relationship between recombination rate and chromosomal position showed an almost exponential shape in the ancestral‐Z, while for the added‐Z the pattern was less clear . Genetic diversity and Tajima's D were in turn significantly higher in the PAR and MREC compared to the NONREC  and repeat proportion  showed a significant difference between these regions . Also, LD measured with D′ showed significantly higher values in added‐Z . The remaining parameters did not differ between the ancestral‐ and added‐Z parts of MREC (p > .05).When the genome characteristics were compared between the ancestral‐ and added‐Z within the MREC, Tajima's D , repeat proportion  and Fay and Wu's H (τ = 0.42 p = 5.4e‐09) correlated positively, while exon proportion  and linkage disequilibrium  correlated negatively, with the recombination rate. The remaining correlations were nonsignificant. When these associations were tested separately for the ancestral‐ and added‐Z . Further, dN/dS was higher in the added‐Z compared to the ancestral‐Z . However, MREC ancestral‐Z had only five genes with remaining W copy, so these results need to be interpreted cautiously. Moreover, after correction the p‐value for multiple testing with Holm's corrections , none of the region comparisons remained statistically significant for genes with W copy . After correction the p‐value f or multiple testing with Holm's corrections , only the results for dS remained statistically significant for genes without W copy  and positively with dS in ancestral‐Z . After correction the p‐value for multiple testing with Holm's corrections , all correlations became statistically nonsignificant also for genes without W copy.When we tested the gene‐specific correlations between the amount of recombination and evolutionary rates (within MREC), none of the correlations were significant for genes with W copy, whereas in genes without W copy the rate correlated negatively with dN/dS in MREC  is not recombining at all in either of the sexes . This creates three distinct regions within the chromosome: the small PAR where both sexes recombine, the male‐recombining Z (MREC) and the nonrecombining Z (NONREC). The nonrecombining region is probably slightly larger (up to 79%) as the two  un‐anchored sex‐linked scaffolds (94 and 4139) most probably originate from this region. In a typical case of heteromorphic sex chromosomes, the chromosome pair does not recombine in the heterogametic sex  have acted on a region with no or very low amount of recombination over a long evolutionary time scale, and thus that restricted recombination in the large central part of the Z chromosome has been a consistent feature over a relatively long period of time in the great reed warbler. The crossing over events cluster at the chromosome ends, where the recombination rate showed further fine‐scale dependence on the chromosomal position by increasing towards the ends. This pattern is in line with the general bias of recombination towards the telomeres in male vertebrates  while in MREC there was no such connection . Further, centromeric regions (which make up part of the NONREC) tend to have lower gene density in general. Perhaps the clearest consequence from the apparent lack of recombination is the loss of genetic diversity. The level of genetic variation is determined by mutations, recombination, genetic drift, selection, GC‐biased gene conversion and demography . The fact that males have a higher mutation rate compared to females . Assuming that the PAR reflects roughly the general autosomal diversity level, the male‐specific Z (comprising the MREC and NONREC) shows a relative diversity of 0.27 compared to the PAR. If purely neutral, a ratio this low would need an unrealistically low NE‐males/NE‐females; too low to be explained by the polygynous mating system  could result from male‐mutation bias in the latter. The difference in relative diversity values between the MREC and NONREC results most likely from the observed lack of recombination and following selection acting on several linked sites (linked selection) in the latter, since male mutation bias, effective population size, drift and demography should affect these two regions similarly.The apparent lack of recombination in the central part of the Z chromosome shapes the evolution of this region drastically compared to the recombining parts, which is manifested as higher linkage disequilibrium and amount of repeats, as well as a lower GC content. This region also had a lower proportion of exonic sequence, but concluding that this is due to gene degeneration should be done cautiously as the higher amount of repeats in the NONREC could partly decrease the proportion of exons in the bins of a certain size. Indeed, exon density decreased with higher repeat density in NONREC , which enhances the effect of selection further, since also recessive alleles are exposed to selection in females. Fay and Wu's H was mostly positive or close to zero within the NONREC , which is not expected if the reduction in diversity had been primarily caused by recent positive selection. Thus, we hypothesize that purifying selection has played an important role in lowering the diversity and causing negative Tajima's D values in this part of the neo‐sex chromosome. However, even in very detailed studies, separating the contribution of different selective processes on linked sites remains challenging Stephan, .There were three intervals within the NONREC that showed high positive Tajima's D values and thus stood out from the general pattern of negative values in this region Figure . Closer Linkage disequilibrium is strongly affected by recombination, which breaks the linkage between loci. Our results were in line with this, with LD being significantly higher in the NONREC and showing strong negative correlation with recombination rate. The positive association between recombination and GC content in the region comparisons was expected too, since crossover events are known to affect the nucleotide composition of sequences through GC‐biased gene conversion  between ancestral‐ and added‐Z, the age difference in the sex‐linkage could have affected the sequence patterns between these regions. Some of the unexpected correlation patterns for the added‐Z, such as the lack of correlation between recombination and GC content, could be caused by the fusion history of this region. As the recombination rate depends on the physical position along the chromosome, the current recombination landscape of the added‐Z could differ from the previous autosomal one and this change could lead to the situation where the GC content reflects both previous autosomal and current recombination rate effects. This could disrupt the sign of ongoing GC biased gene conversion and the following expected positive correlation with recombination rate. Ancestral‐Z had higher repeat proportion, while both MREC regions showed positive correlation between recombination rate and repeats. Thus, the amount of repeats seems to be a complex result of age of sex‐linkage and varying sign of correlation between recombination rate and different repeat types Figure .4.5Zosterops spp.), another Sylvioidea genus that shares the same neo‐sex chromosome system as the great reed warbler, also found that added‐Z genes and autosomal genes had similar dN/dS values despite the lower recombination rate on chromosome Z  showed evolutionary rate variation depending on both the age of sex‐linkage  and the gene expression pattern, highlighting the role of other factors in addition to recombination affecting these rates , but instead our results show nonsignificant association. A similar kind of lack of association has been observed for example in primates  of recombination. Possibly, rare recombination within the NONREC may happen often enough to prevent accumulation of slightly deleterious mutations. The fused added‐Z region seems to reflect previous autosomal and current Z‐linked recombination landscapes in its sequence characteristics. This region showed slightly different recombination landscape compared to the ancestral‐Z, and further research is needed to test if this is due to its gene content and the following differences in selective regimes.Suvi Ponnikas and Bengt Hansson designed the study; Suvi Ponnikas, Max Lundberg and Hanna Sigeman performed analyses, and Suvi Ponnikas wrote the manuscript with input from all coauthors.The authors declare that they have no conflict of interest.SupinfoClick here for additional data file.