Strong declines of grassland species diversity in small and isolated grassland patches have been observed at local and landscape scales. Here, we study how plant–herbivore interaction webs and habitat specialisation of leafhopper communities change with the size of calcareous grassland fragments and landscape connectivity. We surveyed leafhoppers and plants on 14 small (0.1–0.6 ha) and 14 large (1.2–8.8 ha) semi-natural calcareous grassland fragments in Central Germany, differing in isolation from other calcareous grasslands and in the percentage of arable land in the surrounding landscape (from simple to complex landscapes). We quantified weighted trophic links between plants and their phytophagous leafhoppers for each grassland fragment. We found that large and well-connected grassland fragments harboured a high portion of specialist leafhopper species, which in turn yielded low interaction diversity and simple plant-leafhopper food webs. In contrast, small and well-connected fragments exhibited high levels of generalism, leading to higher interaction diversity. In conclusion, food web complexity appeared to be a poor indicator for the management of insect diversity, as it is driven by specialist species, which require high connectivity of large fragments in complex landscapes. We conclude that habitat specialists should be prioritized since generalist species associated with small fragments are also widespread in the surrounding landscape matrix.The online version contains supplementary material available at 10.1007/s00442-020-04830-6. Landscape-scale agricultural intensification often leads to loss, degradation and fragmentation of remaining natural habitats and poses great threats to biodiversity  in contrast to other studies  for each fragment. Relative cover (%) per species was calculated by dividing the cover of the given species by total plant cover plus bare ground cover and litter cover.At the beginning of June 2010, we designated six transects within homogeneous vegetation per fragment for surveying plants and leafhoppers (Hemiptera: Auchenorrhyncha). The transects were well spread across the fragments, about 10–15 m away from each other within a fragment (minimum distance 3 m); fragment edges were avoided. We recorded the cover (%) of vascular plant species, bare ground cover (%) and litter cover (%) of each transect in botanical plots (one 1 × 5 m plot per transect). Subsequently, we calculated the mean relative cover of each species and the total number of plant species  centred on the botanical plots  during dry weather on three occasions . These sweep-net transects exceeded the botanical plots in size and were approximately 10 m long. The specimens of leafhoppers caught were transferred into ethanol (70% vol) and subsequently identified to species level , generality (mean number of plant species per leafhopper species) and interaction diversity (Shannon diversity of interactions) . Of the 67 leafhopper species present in the food webs, 38 species with 2524 specimens were classified as habitat generalist and 29 species with 4182 specimens as habitat specialist (Online Resource 1). 24% of habitat generalist species and 66% of habitat specialist species were monophagous species. The 28 food webs contained altogether 968 plant–herbivore interactions Below is the link to the electronic supplementary material.


Whole fruit increased among children/adolescents , but not adults, while the vegetable contribution was generally unchanged. The proportion of the population below the EAR increased by 23.8% on a relative scale or 9 percentage points on an absolute scale (38.3–47.4%). Declines in vitamin C intake is driven largely by decreases in fruit juice coupled with modest increases in whole fruit. Due to associations between vitamin C intake and numerous health outcomes these trends warrant careful monitoring moving forward.Low intakes of fruits and vegetables have resulted in suboptimal intakes of several micronutrients, including vitamin C. This cross-sectional study used data from 84,902 children/adults (≥1 y) who completed a 24-h dietary recall as part of the United States National Health and Nutrition Examination Survey (1999–2018). Mean vitamin C intakes from foods/beverages were calculated as were trends in major food/beverage sources of vitamin C. Percentages below the Estimated Average Requirement (EAR) were estimated. Overall, mean vitamin C consumption declined by 23% (97–75 mg/d;  Most of the United States (US) population currently fails to meet fruit and vegetables recommendations provided by the most recent Dietary Guidelines for Americans (DGAs) . Recent The health benefits of vitamin C are well documented: vitamin C is essential to normal function of the immune system ,7,8, skiDespite the clear benefits of adequate vitamin C intake this nutrient has consistently been classified as underconsumed in the DGAs since 2005 ,15,16, tThis cross-sectional study was based on data from the 10 most recent 2-year cycles 1999–2018) of the continuous NHANES. NHANES data is a nationally representative survey that uses a complex multistage probability sample to create a representative sample of the noninstitutionalized civilian US population. NHANES is the flagship survey for assessing the diets of the US population. The response rates to the survey differ by year, but were 62.8% in 2017–2018 and 82% in 1999–2000 [ of the cFor primary analyses, dietary data came from a single 24-h dietary recall conducted in-person. A single 24-h recall for a large sample size will lead to an unbiased estimate of population-level average intakes . RespondSecondary analyses examined the proportion of the population consuming vitamin C levels below pre-specified thresholds . Because a single 24-h recall  cannot reliably estimate the population distribution of intakes we used the National Cancer Institute (NCI) method to estimate the proportion of the population consuming intakes below this threshold. As this method requires a subset of participants to have a second 24-h recall we used data from 2003-onwards for these analyses . The second dietary recall is conducted over the telephone after the initial 24-h recall.To examine trends in vitamin C intake stratified by food/beverage source (hereafter referred to as source) data were broken into five mutually exclusive groups: 100% fruit juice, whole fruit, vegetables, fruit drinks and other sources. These categories were created based on preliminary analyses examining the top sources of vitamin C in the diet. Each category was defined based on the prefix code within the Food and Nutrient Database for Dietary Studies . For exaAnalyses were conducted for the total population age ≥1 y who had at least one valid 24-h dietary recall as defined by NCHS staff. Per standard analysis procedures of 24-h dietary recall data, there was no additional exclusion of individuals based on extremely low or high energy intakes. Pregnant and lactating women were included in the analysis of dietary recalls.Secondary analyses compared biochemical indicators of vitamin C measured in sera for 2003–2006 (pooled from two cycles) and 2017–2018 among individuals with valid serum vitamin C levels (age ≥ 6 y) participating in the laboratory assessment of NHANES. Pregnant women were excluded. The protocols for data collection and analyses were generally comparable across the years and included both fasted and unfasted samples ,23. ConsFor each 2-year cycles, the survey-weighted mean and corresponding 95% confidence interval of average vitamin C intakes from sources was calculated. A survey-weighted linear regression model was then used to determine if there was a significant linear trend in intakes of vitamin C. Analyses were repeated for age/sex groups defined by the Dietary Reference Intakes (DRI) publication . AnalyseFor primary analyses, the sample size was 84,902 . Declines were particularly dramatic among children 1–3 y (change from 1999–2000 to 2017–2018: −32.1%), 14–18y females (−39.6%), 14–18y males (−33.6%), women 51–70 y (−30.7%), and men 51–70 y (−25.7%). The only sub-groups not experiencing a statistically significant decline were 9–13y males and females, 31–50 y females and older adults (≥71 y). Because other changes in the composition of the population can affect trends in dietary intakes, sensitivity analyses evaluated overall trends adjusted for age, sex, race/ethnicity and energy intakes. There was no observable difference between adjusted and unadjusted results  were included in the analysis of serum vitamin C levels. The results of the serum vitamin C analysis diet are shown in In this large nationally-representative survey of more than 84,000 participants with high-quality dietary data collected over a 20-year period we observed a 22.6% decrease in vitamin C intakes. This decrease was observed amongst all but a limited number of age/sex groups and appears to be driven by reduced contribution of vitamin C from 100% fruit juice and no marked change in vitamin C from vegetables. Vitamin C from whole fruits increased for children/adolescents, but not adults. From 2003–2004 to 2017–2018, the proportion of individuals with intakes below the EAR increased from 38.3% to 47.4%. Analyses of serum vitamin C observed a non-significant decrease in vitamin C levels, particularly for adults, but the proportional decline was less than was observed for diet.While far from optimal, prior population-based studies have shown that the US-diet has modestly improved over the past 15–20 years. Notable changes include increased whole grain, fiber, polyunsaturated fat and whole fruit consumption and decreased consumption of added sugars and solid fats . Fiber . The generally flat intake of vegetables represents an important public health challenge as vegetables have numerous health benefits and are nutrient dense and energy sparse. Multiple campaigns and policies have been implemented to encourage vegetable consumption but there remain numerous barriers to intake. The high cost of whole fruit and vegetables, particularly fresh versions, is one often-cited barrier [While the changes in vitamin C sources from juice and whole fruit were compelling it is worth noting that we observed no marked change in vitamin C contributions from vegetables, which is consistent with relatively flat consumption of vegetables over this study period. For children, we observed a modest trend towards less vitamin C from vegetables and for adults there was some suggestion of such a trend , but rather on its potential impact on the immune system, skin health and collagen synthesis, nervous system and potentially on reactive oxidative species . On a coStudy limitations include the use of self-reported dietary data which is subject to systematic and random errors . DespiteIn this large population-based study we observed a dramatic decline in vitamin C intakes that appears to be driven by declines in fruit juice consumption. A modest increase in vitamin C from whole fruit could not make up the gap due to declines in 100% fruit juice consumption. While at the population-level there is little risk of severe vitamin C deficiency, given observed relationships and associations between vitamin C intake and numerous health outcomes these trends are of potential concern. The proportion of individuals with vitamin C intakes less than the EAR appear to be greater than for other nutrients of public health concern including calcium (for men) and potassium. There is tremendous need to increase fruit and vegetable consumption in the United States and multiple approaches will likely be necessary to impart these changes. Looking forward, vitamin C intakes should be continually monitored and identifying specific sub-populations at greater risk for inadequate vitamin C intakes could be identified. Close examinations of dietary trends for specific dietary constituents of interest beyond the overall topline trend can help us understand why the diet may be changing and identify potential strategies to improve diet.


Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes. Pancreatic cancer is commonly characterized by extensive desmoplastic stroma and an environment that is poorly supportive of adaptive immune responses, yet like many other cancers, the degree of T cell infiltrate in pancreatic tumors is correlated with patient outcome –4. T celQuantitative immunohistochemistry (IHC) has long represented the gold standard by which tumor infiltrating immune populations can be assessed, and recent advances in multispectral IHC combined with automated image analysis have made possible an unprecedented ability to map out the immune environment of tumors. However, these approaches are limited by the availability and quality of antibodies, and complex multispectral panels require extensive validation to confirm the specificity and selectivity of binding. Recently, multiple groups have shown that the quantity of a diverse array of infiltrating immune cell types in a specimen can be inferred based on characteristic gene expression patterns unique to or enriched in specific cell types , 6. In t+ T cells from a combination of CD4+, CD8+, and other cell types that express CD3. This aggregation may overcome the limitation of low T cell-derived RNA transcripts in poorly-immune infiltrated tumors. As an alternative, we identified gene signatures that were enriched in highly T cell infiltrated tumors that are associated with increased disease-free survival in other patient cohorts. These data demonstrate that immune infiltration remains an important predictor of outcome in pancreatic cancer patients, and that RNA analysis can provide an important addition to IHC data to understand the complexities of the immune environment that influence patient outcome.Thus far we are not aware of any studies that have directly compared IHC quantification of immune infiltration to RNA-Seq-based analyses in pancreatic cancer. In this study we aim to directly compare conventional IHC and gene expression-based approaches to characterize the immune environment of pancreatic cancer. We hypothesize that RNA-Seq analysis can provide a comprehensive alternative to quantitative IHC for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of 39 pancreatic adenocarcinoma patient tumors with matched quantitative IHC, and evaluated approaches to quantify infiltrating immune cells using gene expression data. We found limited agreement between IHC and RNA-Seq analysis of infiltrating cells, however concordance was greatest when multiple cell types were aggregated to identify a mixed population, such as CD3+, CD68+, and CD8+ cells as previously described [2 tissue for each patient. Primary outcome was overall survival.We conducted a prospective cohort study of resectable pancreatic masses to determine which immunologic parameters have prognostic value. All procedures were approved under Providence Portland Medical Center Institutional Review Board, with approval number IRB 10–037, and patients provided written informed consent. We restricted our analysis to adult patients who underwent surgical resection for pancreatic masses. Patients were recruited from June 2010 to November 2014 at Providence Portland Medical Center in Portland, OR, where the research was conducted. Inclusion criteria included patients 18 years or older who had a diagnosis of a pancreatic or ampullary mass who were scheduled for surgical resection. Patients had to be able to give informed consent and could not have a diagnosis of a prior malignancy unless they were disease free for 10 years. We included patients who were subsequently determined to have other histologies. Demographics and survival of these patients are outlined in Of the patients above with pancreatic masses, we randomly selected 39 patients with pathologically diagnosed pancreatic adenocarcinoma with no neoadjuvant treatment and with matched quantitative IHC for RNASeq. All subsequent analyses of IHC and RNASeq were performed on unpretreated patients. A representative Hematoxylin and Eosin (H&E) stained slide for each formalin-fixed paraffin-embedded (FFPE) tissue block specimen was reviewed by a board-certified pathologist for tumor content and tumor-rich regions were identified for microdissection. Blocks were matched but not in series with IHC sections. 5 μm thick unstained sections on glass slides were processed for DNA and RNA purification by the Providence Molecular Genomics Laboratory. The FFPE tissue sections were deparaffinized using Envirene (Hardy Diagnostics) followed by RNA extraction and purification using the Qiagen AllPrep DNA/RNA FFPE kit. 85ng of input RNA was used to prepare sequencing libraries using the Illumina TruSeq RNA Exome kit. Sequencing of the RNA Exome libraries was performed on the Illumina HiSeq 4000 instrument at 2 x 75 read paired end configuration. Transcripts were quantified using salmon-v.0.11.2 [cell type enrichment analysis from gene expression data for 64 immune and stroma cell types, whereas CIBERSORT provides absolute and relative abundance of different immune cell types depending on the specified gene set. In the present analysis, the LM22 signature provided by CIBERSORT was applied. We also applied EPIC (10) and MCPcounter (11) to estimate the abundance of immune cell population. EPIC estimates the proportions of Immune and Cancer cells by using RNA-Seq-based gene expression reference profiles from immune cells and other nonmalignant cell types found in tumors. MCPcounter quantifies abundance of tissue-infiltrating eight immune and two stromal cell populations based on transcriptome profile.RNA-Seq-based cell type deconvolution was performed using xCELL (5) and CIBERSORT (6), with TPM gene expression levels as input. xCELL was used to perform Initial clustering of patients based on infiltrating cell types was performed using ClusterVis . Princip+ and CD8+ T cells according to quantitative IHC and sub-categorized into those with high infiltration of both populations (CD3HICD8HI) versus low infiltration of both populations (CD3LOCD8LO). Using RNA-Seq data from these patients, gene expression analyses were performed using a univariate two-sample T-test with a stringent false-positive threshold to identify genes significantly differentially expressed (p<0.001) in our patients [http://xcell.ucsf.edu.Patients were categorized into high and low infiltration of CD3patients , 13. Thepatients . These gpatients  as a valpatients , 17 wherSurvival data were analyzed using Prism . Overall survival of groups was compared using a log rank test for differences in Kaplan-Meier survival curves. All cutoffs for high/low infiltration by RNA analysis use median values. Gene expression analyses were performed using a univariate two-sample T-test with a stringent false-positive threshold to identify genes significantly differentially expressed (p<0.001) , 13. Cor+ T cell infiltrates in surgically resected pancreatic adenocarcinoma correlate with improved outcomes using a Cox proportional hazards model, and remained prognostic by multivariable analysis [+ T cell infiltrates and CD68+ macrophage infiltrates did not correlate with outcome [+, CD8+ and CD68+ cell numbers in tumors, we were able to identify cutoffs that demonstrated that patients with high CD3+ T cells or high CD8+ T cells exhibited improved survival, but again CD68+ macrophages did not correlate with survival ; CD8+ equivalent (CD8); and CD68+ macrophage equivalent (MO+M1+M2). We then evaluated the correlation between infiltration determined by CIBERSORT analysis of RNA-Seq versus quantified immunohistology. Both CD3+ and CD8+ T cells showed a weakly positive correlation between the IHC and CIBERSORT assessments, but CD68 did not correlate well between histology and CIBERSORT , but the CD8 and macrophage scores were not able to discriminate patients with significantly different overall survival , and examined the correlation of each cell type with the expression of genes in our panel. To find potential patterns of biological significance, we correlated the expression of the gene set with the infiltration of immune cells across the TCGA cohort and performed clustering to gather co-regulated genes and cells together. We discovered that as with our cohort, there was a strong correlation between infiltrating T cells and dendritic cells in pancreatic tumors, and these correlated closely with the expression of genes in our panel Click here for additional data file.S2 FigRows are centered; no scaling is applied to rows. Both rows and columns are clustered using Manhattan distance and average linkage.(PDF)Click here for additional data file.S3 FigRows are centered; no scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage.(PDF)Click here for additional data file.S4 Figvs. IHC CD3+, ii) MCPcounter CD8 T cell vs IHC CD8+, and iii) MCPcounter monocytic lineage vs IHC CD68+ cell populations from the same patient. Each symbol represents one patient. b) analysis as in a) for i) EPIC Bref CD8 T cells and ii) EPIC Tref macrophages. c) Pearson correlation matrix of infiltrating T cell subsets calculated from RNASeq data using xCELL, CIBERSORT, MCPcounter, EPIC Bref, and EPIC Tref. Rows are centered; no scaling is applied to rows. Both rows and columns are clustered using Manhattan distance and average linkage.a) The immune infiltrate score determined by MCPcounter of i) MCPcounter T cell (PDF)Click here for additional data file.S1 Table(TXT)Click here for additional data file.


Correlation with age was significant (p < 0.05) for PDFF of the sacrum (men (m): r = 0.58; women (w): r = 0.54), L5 , the gluteal  and paraspinal  muscles in both genders. BMI correlated significantly with the paraspinal musculature in men (r = 0.46) and women (r = 0.33). Correlation testing revealed significant correlations (p < 0.05) between the two osseous  and the muscle compartments  in both genders. Bone marrow and muscle fat infiltration patterns were not significantly associated with each other at the sacral and lower lumbar spine region. The presented data suggest that the two compartments may have distinct pathophysiological fat infiltration patterns. However, further clinical studies are needed to support the results.This analysis investigated the age- and BMI-related variations of fat distribution in sacral and lumbar bone marrow and their association with local muscle fat content in order to detect fat distribution patterns and variations in healthy adults using proton density fat fraction (PDFF) measurements. A six-echo 3D spoiled gradient-echo sequence was used for chemical shift encoding-based water-fat separation at the sacral and lower lumbar region in 103 healthy volunteers. PDFF values of the sacrum, 5 It is known that in diseases like osteoporosis or sarcopenia fat content of bone marrow is elevated and the fatty infiltration of muscular tissue is increased3. As osteoporotic fracture risk significantly increases due to aging, understanding the pathophysiology and subsequent structural alterations accompanying muscle loss and increased bone marrow adiposity is of high scientific but also of substantial clinical and economic value5. Besides aging, an unfavorable body composition has been shown to be associated with bone marrow composition, bone health and fracture risk7. Against the background of an aging population in context of the demographic shift and both obesity and undernutrition being growing health problems worldwide, exploring the connection of structural changes in muscular tissue and bone marrow and their interactions with each other will gain importance in the near future.In senescence macroscopic changes in regard of bone marrow adiposity and muscular fatty infiltration as well as decreasing unsaturation and increasing saturation levels of fatty acids have been observed8. As shown in previous studies, bone marrow PDFF values show specific distribution patterns inter- and also intra-individually, depending on the topography within the lumbar spine, with increasing PDFF from cranial to caudal (L1 to L5)11. Furthermore, an age-dependent correlation between the structural composition of the local lumbar musculature and vertebral bone marrow fat has been described recently12. A systematic analysis of the functional impact associated with these pathophysiological processes using isometric strength measurements showed that an increased fatty infiltration of the paraspinal muscles significantly correlated with reduced muscle strength14. Moreover, several recent studies elucidated the connection between a sedentary lifestyle, muscle volume loss and increasing intramuscular fat, decreasing BMD and associated evolving facture risk and delayed bone healing processes16. But not only fracture risk and muscle strength, the latter being connected to frailty in elderly persons, are associated with bone marrow composition and local muscle fat content. In previous studies ectopic, intramuscular fat accumulation has been shown to be associated with decreasing insulin sensitivity and metabolic diseases18. Furthermore, vertebral bone marrow fat content has also been shown to be correlated with insulin resistance in postmenopausal women diagnosed with Type 2 Diabetes, which suggests the use of vertebral PDFF for screening of obese subjects with a tendency towards developing metabolic syndrome19.But what is the immanent clinical use of evaluating and comparing ectopic fat distribution patterns in the paraspinal and gluteal musculature and the fat content of the vertebral bone marrow in a healthy study collective besides elucidating the interplay of osseous and adjacent muscle tissue on a fundamental level? First, vertebral bone marrow fat fraction has previously been shown to increase fracture discrimination power when used with bone mineral density (BMD)21. This allows for calculating surrogate values like the proton density fat fraction (PDFF), and with MRS also the fat (un)saturation levels23.Structural composition of vertebral bone marrow and muscle can be assessed using magnetic resonance spectroscopy (MRS) and chemical shift encoding-based water–fat magnetic resonance imaging (MRI)25. However, the age- and BMI-related variations of the sacral bone marrow composition and their association with local muscle fat content have not been investigated yet.Non-invasive muscle status assessment with chemical shift encoding-based water-fat MRI would allow for early interventions in subjects showing local fat accumulation levels. With obesity, osteoporosis, their associated diseases and ageing populations being on the rise, an accurate assessment tool identifying patients with abnormal values would be of benefit for clinicians in the futureThus, the purpose of this analysis was to investigate lower lumbar and sacral bone marrow fat content based on chemical shift encoding-based water-fat MRI in men and women and their correlation with the composition of local musculature and analyze compositional variations with regard to age and BMI.26. The study protocol and procedures were approved by the ethical committee of the Faculty of Medicine of the Technical University of Munich, Germany. Subjects were screened for eligibility and included if age was equal to or greater than 18 years, they had no history of severe diseases or surgery within the last three months, acute physical impairment or standard contraindications for MRI. All subjects gave written informed consent prior to study inclusion. All subjects underwent an MRI including a 3D spoiled gradient echo sequence at the upper pelvic/ gluteal region. On the basis of the acquired sequence the measurement of PDFF values was conducted.103 volunteers (72 women and 31 men) were recruited at the Institute for Nutritional Medicine, Klinikum rechts der Isar, Technical University of Munich, from a large observational study aiming at evaluating determinants of basal metabolic rate3, acquisition matrix size = 268 × 200, acquisition voxel size = 1.5 × 1.5 × 1.5 mm³, SENSE with reduction factor = 2.5 × 1.0, receiver bandwidth = 1413 Hz/pixel, 2 averages, scan time = 2 min 1 s28.All subjects underwent MRI at 3T . Subjects were positioned head-first in a supine position. An axial six-echo multi-echo 3D spoiled gradient-echo sequence was applied for chemical shift-encoding based water-fat separation using anterior and posterior coil arrays. The sequence acquired the six echoes in a single TR using non-flyback (bipolar) read-out gradients and covered the gluteal region with the following imaging parameters: TR/TE1/ΔTE = 8.2/1.24/1.0 ms, flip angle = 5°, FOV = 400 × 300 × 140 mmet al.29. Segmentations were performed by a radiologist using the free open-source software Medical Imaging Interaction Toolkit . The lateral masses of the sacrum, the fifth lumbar vertebral body (L5), the gluteus maximus muscles and the paraspinal muscles were included in the analysis and manually segmented bilaterally in the PDFF maps in 5 slices at the height of L5 and the first sacral vertebral body level, respectively. At the level of L5 the region of interest (ROI) was placed in the center of the vertebral body, thus avoiding the accidental inclusion of Modic endplate changes. PDFF values of each resulting ROI were recorded and the average value of both sides was calculated. Figure The gradient echo imaging data were processed online using the fat quantification routine of the MR vendor . PDFF maps were generated using a complex-based water-fat separation algorithm that accounts for known confounding factors including a single T2* correction, phase error correction and the consideration of the spectral complexity of fat using the multi-peak fat spectrum model of Ren 2).Height was measured without shoes in a standing position using a stadiometer , and reported to the nearest 0.5 cm. Weight was assessed on the day of the MRI scan. BMI was calculated as the quotient of weight in kg and height in meter squared  from the study population were used to determine the reproducibility of the PDFF measurements. All bone marrow and muscle segmentations as outlined above were performed a second time by the same radiologist 8 weeks after the initial segmentations. Intra-reader reproducibility error was expressed as root mean square absolute precision error in percent (absolute units) according to Gluer All statistical tests were performed using SPSS . All tests were performed using a two-sided 0.05 level of significance. Graphpad Prism Version 7  was used for generating Fig. All procedures performed in this study using human participants were in accordance with the ethical standards with the institutional and the 1964 Helsinki declaration and its later amendments.p = 0.980), neither was BMI   in men (11.1 ± 10.7%) than in women (12.9 ± 9.8%). PDFF of the paraspinal muscles showed significant differences between men (14.2 ± 7.8%) and women (25.9 ± 9.3%) (p < 0.0001). No significant differences between men and women could be detected in PDFF values of the sacrum (p = 0.903), vertebral body L5 (p = 0.741) and the gluteal muscles (p = 0.167) , L5  and the paraspinal muscles  . BMI correlated significantly with the PDFF measurements in paraspinal muscles  in both genders (Table Correlation with age was significant for the PDFF measurements of the sacrum (men: 1) Table . PDFF ofp < 0.05) only between the two osseous compartments  in both genders, but not between the gluteal and paraspinal muscle compartments as well as the bone marrow and muscle compartments (p > 0.05).Partial correlation testing with age and BMI as control variables revealed significant correlations  was shown by several groups in the past40. In the present analysis, the paraspinal muscle PDFF correlated with BMI, which points in the same direction. Adding different physiological and ectopic fat depots to a multimodal and multiparametric assessment protocol enables the clinician to identify the obese phenotype, evaluate the individual risk of (obese) subjects more accurately and to initiate early interventions. Against the background of an increasing awareness of sarcopenic obesity leading to loss of muscle mass and functional impairment in the elderly, the identification of subjects at risk is of high clinical importance43, and deeper knowledge about the processes altering bone marrow and muscular tissue composition and their relationship might prove useful for this.Finally, bone marrow fat content and fatty infiltration of the paraspinal musculature have been shown to correlate with subcutaneous and visceral fat depots, which are well known to boost the risk of metabolic disturbancesA possible field of clinical implementation might be adding the presented sequence with a comparably short scan time of two minutes to the scanning protocol of elderly patients at risk for obesity or sarcopenic obesity. Elevated levels of bone marrow and intramuscular PDFF might add quantitative information to the holistic and multi-parametric fracture risk or metabolic syndrome risk assessment. In the light of the potential clinical implementation in primary and secondary prevention the reported data might serve as reference standard for pathology delineation.et al.44. Lastly, the analysis lacks information about potential confounding factors for bone marrow and muscle composition such as physical activity, nutrition or smoking habits. Future analyses should include these potential confounders.There are certain inherent strengths and methodological limitations regarding the design of the present analysis, which are elucidated in the following. First, the large cohort size provides a good basis for age- and BMI-dependent analysis of PDFF variations. However, the men to women ratio as well as the different age groups could be balanced more equally in future studies, as elderly female subjects and male subjects in general are underrepresented. Second, we performed this study with a cross-sectional design. Thus, no intraindividual dynamics over a certain time period can be evaluated. Longitudinal studies with at least one follow-up scan or several scans at defined time points should be concepted to further investigate the temporal dynamics of PDFF changes in individuals. Third, no BMD measurements were performed. However, the reciprocal connection of BMD and PDFF values has been shown by previous studies, amongst others by Kühn In summary, this analysis showed that variations in sacral and lumbar bone marrow composition detected by using chemical shift encoding-based water-fat MRI were age-related, while only the paraspinal muscle fat content correlated with BMI in both genders. However, bone marrow and muscle fat were not significantly associated with each other at the sacral and lower lumbar spine level. In conclusion, this might point to different underlying pathophysiological fat infiltration patterns within the different compartments, but further clinical studies are needed to give this theory more evidence and exclude confounding aspects like smoking habits or level of physical activity.


We also identify differentially expressed genes, provide an initial comparison of genes affected in each genotype and tissue, and provide the pipeline used to analyze the data. Further analysis of this dataset can be used to gain insight into the effects of DGT on various root developmental pathways in tomato.Cyclophilin A/DIAGEOTROPICA (DGT) has been linked to auxin-regulated development in tomato and appears to affect multiple developmental pathways. Loss of DGT function results in a pleiotropic phenotype that is strongest in the roots, including shortened roots with no lateral branching. Here, we present an RNA-Seq dataset comparing the gene expression profiles of wildtype (‘Ailsa Craig’) and Solanum lycopersicum) cyclophilin DIAGEOTROPICA (DGT) has been linked to auxin-regulated development through identification of the gene affected by thediageotropica (dgt) mutation2. Tomatodgt mutants are auxin-resistant and display a pleiotropic phenotype that includes slow gravitropic response, lack of lateral root initiation, altered vascular development, reduced ethylene production in response to auxin, reduced apical dominance, impaired shoot and root growth, reduced fertility, and impaired fruit growth11. DGT likely interacts with auxin transport and signaling in a complex manner. For instance, DGT has been shown to negatively regulate auxin efflux via PIN-FORMED (PIN) transporters by altering subcellular localization of PINs and expression of somePIN genes9.DGT, in turn, is downregulated by auxin at the root tip8, suggesting functional feedback between DGT, auxin, and PINs. Through targeted gene expression quantification with RT-PCR and northern blots, DGT has been demonstrated to affect expression of a number of other auxin-related genes in addition toPIN genes. Thedgt mutation reduces auxin-induced expression of genes encoding certain 1-aminocyclopropane-1-carboxylic acid synthases , SMALL AUXIN UPREGULATED RNA (SAUR) genes, and several members of the auxin-regulatedAux/IAA gene family in a tissue- and developmental stage-specific manner14. However, full transcriptomic profiling of tomatodgt mutants in different developmental zones has not been performed. Given the complex role of DGT in auxin pathways, an exploration into the widespread effects of DGT on the transcriptome may provide valuable insights into its potentially extensive and multifaceted role in development.The tomato  anddgt tomato plants. The root tip provides an excellent system for studying development-related plant gene expression because cell division, elongation, and maturation are not only temporally but also spatially separated in this growth region; this allows for anatomical dissection and analysis of specific developmental zones, such as the meristem, elongation zone, and differentiation zone. The root tip is also most appropriate for this study because thedgt phenotype is the strongest in the root tip and has been characterized morphologically in the root tip8. We have previously performed histological analyses of tomato root tips including the meristem, elongation zone, and maturation zone, and demonstrated a decrease in length and number of cells of thedgt meristem and elongation zone8, whereas the initiation of lateral root primordia was abolished in thedgt root maturation zone9. Here, we present an RNA-Seq dataset containing raw reads and abundance estimates for three replicates in each zone and genotype, the pipeline used for analysis, and an initial exploration of expressed and differentially expressed genes in each developmental region that can be used to guide future investigations.In this study, we perform a global analysis of gene expression inSolanum lycopersicum) wildtype (WT) and1-1dgt plants in the ‘Ailsa Craig’ background7 were used. Seeds were sterilized in 20% commercial bleach for 30 min and rinsed four times for 10 min with sterile water. Sterilized seeds were vernalized at 4°C for two days to ensure even germination and then planted on media containing 0.2× Murashige and Skoog basal medium with vitamins (PhytoTechnology), 1% sucrose, 10 mM MES buffer pH 5.7, and 0.8% agar. Seeds were germinated in Magenta boxes (16 seeds per box) in a growth chamber at 21°C under long day  conditions and light intensity as in Ivanchenkoet al. (2013). Root samples were dissected under DIC optics at 4x objective as in Ivanchenkoet al. (2006). On average, 50–100 root portions were collected per biological replicate. The meristem was dissected between the root tip and the root portion where tissue becomes more transparent. The elongation zone was collected from the proximal meristem border and the first hair bulge, and approximately 1 cm portions were collected above the first hair bulge for the differentiation zone.Three biological replicates for each tissue and genotype were performed. Seven-day old tomato (Life Technologies) on ice, and total RNA was prepared using the RNeasy Mini kit (Qiagen) according to manufacturer’s recommendations. The RNA pellets were dried in 1.7 mL centrifuge tubes and solubilized in 178 µL 1X RNA Secure Reagent (Ambion) preheated at 65°C, and incubated at 65°C for 10 min, mixing by pipetting a few times. Then 20 µL 10X DNase I buffer and 2 µL RNase-free DNase I (Ambion) were added to each tube, and tubes incubated for 10 min at 37°C. 700 µL RLT buffer was added (to which 7 µL 2-Mercaptoethanol was freshly added) to each sample, which were then mixed by vortexing. 500 µL ethanol was then added and samples were mixed again by vortexing. Each sample (2 X 700 µl) was applied to an RNeasy Mini spin column from the RNeasy kit and RNA cleanup performed following the manufacturer’s instructions. Each sample was eluted with 30 µL nuclease-free ultrapure H2O, and the RNA concentrations were measure using a NanoDrop 1000 spectrophotometer (Thermo Fisher).Tissue samples were collected in Plant RNA Reagent (Illumina) and sequenced as single-end 51 bp reads on the Illumina HiSeq 2000 using a total of two lanes.RNA-Seq library preparation and sequencing were performed at the Oregon State University Center for Genome Research and Biocomputing. Libraries were prepared using the TruSeq RNA Library Prep Kit v2  rsem-prepare-reference function. Using RSEM, raw sequence reads were then aligned to the reference transcript sequences and estimated transcripts per million (TPM) values were calculated using default parameters. Resulting gene-level estimates from biological replicates were merged into a single input matrix andEBSeq17 (version 1.26.0) was then used to test for differential expression betweendgt and WT for each root-tip zone. Raw RNA-Seq reads, abundance estimates, and differential expression analysis are publicly available in theSequence Read Archive (SRA) andGene Expression Omnibus (GEO), seeData availability.Sequencing produced 5-8 FASTQ files per replicate, which were merged into a single FASTQ file per replicate. Reference transcripts were extracted and preprocessed from the NCBI18). Following EBSeq analysis, genes were filtered for PPDE = 1 to identify those which were differentially expressed. Of the differentially expressed genes, those with postFC (dgt over WT) > 2 were considered upregulated indgt and those with postFC < 0.5 were considered downregulated indgt. Upregulated and downregulated genes were compared between zones and genotypes (19). Principal component analysis (PCA) of the data was performed using TPM values of each gene and was calculated using the scikit-learn20 PCA function with default parameters  and posterior fold change (postFC). Genes with an average of TPM > 2 across biological replicates were compared between zones and genotypes  the DGT protein was shown to hold a conserved auxin signalling function down to moss, but it is not mentioned here; and 2) other immunophilins present overlapping roles with DGT, a paradigm worth mentioning somewhere in the introduction.Are sufficient details of methods and materials provided to allow replication by others?YesIs the rationale for creating the dataset(s) clearly described?YesAre the datasets clearly presented in a useable and accessible format?YesAre the protocols appropriate and is the work technically sound?YesReviewer Expertise:Plant BiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. “RNA-Seq analysis of genes affected by Cyclophilin A/DIAGEOTROPICA (DGT) in tomato root development” presents the results of RNAseq analysis of the roots of a tomato mutant affected in Cyclophilin A/DIAGEOTROPICA (DGT) vs wild type. The authors compare three developmental zones in the root tip of mutant and wild type seedlings and identify differentially expressed genes. Considering the pleiotropic effect of the mutation and its relation to auxin signaling and transport, this work is potentially important to understand abnormalities in the root development in thedgt mutant and the role ofDGT in root development. Considering that the formation of lateral root primordia is impaired indgt mutants, this analysis can also be useful in the identification of candidate genes involved in lateral root initiation.The article by Maria Ivanchenko All the raw data are publicly available, the same as the scripts used for the RNA-seq analysis. A minor suggestion: the authors should explicitly state whether any pre-processing steps were performed on the raw sequencing reads, such as removal of low-quality bases, adapter filtering, or trimming. Regarding the PCA analysis, one of the WT replicates does not group closely with the other WT samples. It could be useful if the authors provide some insight on why this occurs and discuss the effect of this on TPM variation among replicates.Are sufficient details of methods and materials provided to allow replication by others?PartlyIs the rationale for creating the dataset(s) clearly described?YesAre the datasets clearly presented in a useable and accessible format?YesAre the protocols appropriate and is the work technically sound?YesReviewer Expertise:JD: Plant development, root development, root growth and branching, lateral root development. GR-A: Transcriptomics and evolutionary biology.We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


A small percentage of patients with skin infections later develop necrotizing fasciitis (NF). Diagnostic testing is needed to identify patients with skin infections at low risk of NF who could be discharged from the emergency department (ED) after antibiotic initiation. Elevated lactate has been associated with NF; existing estimates of the frequency of NF are based on retrospective reviews, and cases often lack testing for lactate. We present the incidence of patients with skin infections who developed NF and their baseline lactates.In four phase-3 trials, 2883 adults with complicated or acute bacterial skin and skin structure infections were randomized to dalbavancin or comparator, with early and late follow-up visits through Day 28. We prospectively collected baseline plasma lactates in one trial to assess an association with NF.NF was diagnosed in 3/2883 patients (0.1%); all three survived. In the study with prospectively collected baseline lactates (n = 622), 15/622 (2.4%) had a lactate ≥4 millimoles per liter (mmol/L), including 3/622 (0.5%) with a lactate ≥7 mmol/L. NF was not seen in patients with a lactate <4 mmol/L; NF was seen in 1/15 (6.7%) with a lactate ≥4 mmol/L, including 1/3 (33.3%) with lactate ≥7 mmol/L.NF incidence within 72 hours of antibiotic initiation in patients with complicated or acute bacterial skin and skin structure infections was extremely low (0.1%) and occurred in 6.7% with a lactate ≥4 mmol/L. Lactate <4 mmol/L can be used to identify patients at low risk of NF who could be safely discharged from the ED after antibiotic initiation. Streptococcus pyogenes, followed by methicillin-resistant Staphylococcus aureus (MRSA), and can occur in persons without underlying conditions.S. pyogenes, the initial lesion may be mildly erythematous and swollen but progress to extensive inflammation over the next 24 to 72 hours with the skin appearing as dusky, then purplish with bullae, crepitus and/or subcutaneous gas, and with an associated mortality of 30–80%.3A small percentage of patients with serious skin infections later develop life-threatening necrotizing fasciitis (NF). NF has an annual incidence ranging from 0.3–15.5 cases per 100,000 population.Since the initial clinical presentation may be benign and not suggestive of NF, a common clinical question in the emergency department (ED) is whether patients with skin infection can be discharged after antibiotic initiation, or if they may be at higher risk of NF and therefore require a brief hospital admission for observation while receiving intravenous (IV) antibiotic therapy. Adjunctive diagnostic testing is therefore needed to triage patients with skin infections at low risk of NF, who could potentially be safely discharged from the ED. Elevated lactate has been associated with NF and mortality (lactate level >2 or ≥6 millimoles per liter [mmol/L]),Dalbavancin is a long-acting lipoglycopeptide antibiotic approved by the US Food and Drug Administration and European Medicines Agency as a single- and two-dose treatment for adults with acute bacterial skin and skin structure infections (ABSSSI) caused by susceptible Gram-positive organisms, including MRSA.In this analysis, we present the incidence of patients initially presenting with skin and skin structure infections (SSSI) who later developed NF, with available baseline lactate levels from four global phase-3 clinical trials of dalbavancin in SSSI, including 386 patients treated completely in the outpatient setting.11Clinicaltrials.gov),This analysis evaluated data from four phase-3, double-blind, placebo-controlled multicenter trials of 2883 adults with complicated (cSSSI) or ABSSSI. Detailed methods for those trials have been described previously; patients were randomized to receive dalbavancin or linezolid , who could be discharged from the emergency department (ED) after antibiotics.What was the research question?We prospectively collected baseline lactates in patients presenting with skin infections, to assess an association with NF.What was the major finding of the study?Lactate <4 millimoles per liter can identify patients at low risk of NF, who could be safely discharged from the ED after antibiotic initiation.How does this improve population health?A lactate level in the ED could identify patients at low risk of NF, to avoid unnecessary hospital admissions.or surgical site or traumatic wound infection or cellulitis) were enrolled into the trials. Patients must have presented with at least one of the following systemic signs of infection: an elevated body temperature or increased white blood cell count or white blood differential count with ≥10% band forms. In addition to erythema, at least two of the following signs of ABSSSI were required: purulent drainage/discharge; fluctuance; heat/localized warmth; tenderness to palpation; or swelling/induration.Patients ≥18 years of age with cSSSI or ABSSSI involving deeper soft tissue or needing significant surgical intervention  and/or if they developed NF. If signs or symptoms of NF were observed at the prior site of skin and soft tissue infection after enrollment, the treating physicians and surgical team established the diagnosis by surgical exploration in the operating room.We used sensitivity, specificity, positive predictive value, and negative predictive value to determine the predictive value of lactate in determining NF.13S. pyogenes isolated from intraoperative specimens at the site of infection.NF was diagnosed in 3/2883 patients (0.1%) within 72 hours of presentation for skin infection; all three had surgical debridement and survived . ClinicaOur findings suggest that initial lactate levels <4 mmol/L may identify SSSI patients at low risk of NF. The high negative predictive value of 100% for a lactate <4 mmol/L may be relevant for ruling out NF, since no patient from our studies with available lactate levels developed NF with a baseline lactate <4 mmol/L. Our results also support an association between elevated lactate levels and NF, as previously reported.A limitation of this analysis is that lactate levels were not available in all 2883 patients enrolled in the four trials; rather, they were available in patients enrolled in the most recent clinical trial, DUR001-303, where baseline lactates were required by the protocol. This limitation may have been unavoidable as the DUR001-303 protocol was designed to collect baseline lactates in all patients after the observation of a high baseline lactate in the NF patient from a prior study (DUR001-302) who had a serum sample retrospectively tested with value of 4.4 mmol/L. The number of baseline samples tested in the most recent study (n = 622) is robust and allows the calculation of a meaningful sensitivity (100%), specificity (97.8%), and negative predictive value (100%) for lactate as a predictor of NF; the low prevalence of NF may be a limitation in the interpretation of NPV and PPV. Due to the small number of cases of NF, it may not be possible to draw definite conclusions, and larger analyses with more cases of NF may be useful to confirm the association.Overall, the incidence of necrotizing fasciitis within 72 hours of antibiotic initiation in cSSSI or ABSSSI patients was extremely low (0.1%). Patients with a lactate ≥4 mmol/L may be considered at significantly higher risk for NF. Lactate levels <4 mmol/L may identify cSSSI or ABSSSI patients at low risk of NF, who could be safely discharged from the ED after antibiotic initiation with careful follow-up. Further investigation of initial lactate levels as a predictor of NF risk are needed in patients presenting with SSSI.


To investigate the aetiopathology of recurrent epiphora or stickiness after dacryocystorhinostomy (DCR) surgery, identifiable on dacryocystography (DCG), and to assess the success rates of secondary corrective surgeries.Consecutive post-DCR DCG images from patients with recurrent symptoms were reviewed between 2012 and 2015.One hundred fifty-nine eyes of 137 patients were evaluated. Fifty-eight DCGs showed normal postoperative findings, 4 an upper/lower canalicular block, 13 a common canalicular block, 31 a completely closed anastomosis, 50 a narrow anastomosis, and 3 an anastomosis draining into a nasal sinus.The most successful corrective procedures for each failure category were: Lester Jones Tube (LJT) for a normal post-operative DCG (17/18 success), Sisler trephination with tubes for upper/lower canalicular block (1/2 success), redo-DCR with tube for common canalicular blockage (5/6 success), redo-DCR +/− tube for completely closed anastomosis (12/16 success), LJT followed by redo-DCR +/− tube for narrow surgical anastomosis (1/1 and 17/27 success respectively), and redo-external-DCR with tube for anastomosis into a nasal sinus (1/1 success). Redo-DCR was ineffective in patients who had good post-DCR anatomical patency (22% success).This is the first study to report success rates of redo-DCR surgery according to anatomical findings confirmed by DCG. The outcome flow diagram help clinicians recommend procedures that are most likely to be successful for their patient’s specific anatomical abnormality. It also provides a visual tool for the shared decision-making process. Notably, symptomatic patients with a normal DCG post DCR are unlikely to benefit from redo-DCR, with a LJT being the recommended next step.The online version contains supplementary material available at 10.1186/s12886-021-01869-8. The aim of Dacryocystorhinostomy (DCR) surgery is to form an anastomosis between the lacrimal sac and nasal space facilitating tear drainage. Primary DCR surgery has a published failure rate of 3–13% . AlthougIn the authors’ department, dacryocystography (DCG) is routinely performed on patients who report recurrence of symptoms following DCR surgery to visualise the postoperative anatomical flow pathway. Although it is possible to identify the cause of failure based on clinical examination findings, DCG is also useful in identifying the anatomical abnormality of nasolacrimal systems , 10 and The primary aim of this study was to investigate the potential causes of recurrent epiphora and stickiness following DCR surgery, identifiable on DCG imaging. The secondary aim of this study was to establish the outcomes for subsequent revision surgery. This study intends to provide guidance for the decision-making process for patients experiencing failure of DCR surgery by providing surgical success rates of subsequent revision surgery according the underlying cause of failure.This study was conducted in full compliance with the declaration of Helsinki. Permission to perform this study was granted by Moorfields Eye Hospital NHS Foundation Trust Audit committee review board (reference number: CA16/AD/19).The Moorfields Eye Hospital lacrimal clinic is a tertiary referral center for complex lacrimal disease. Consecutive patients experiencing failure after DCR from 2012 to 2015 were reviewed. A lacrimal consultant reviewed DCG images and identified any DCG abnormalities (DE). Clinical data were collected from electronic and paper hospital records. Symptomatic success after lacrimal surgery was classified as the patient being subjectively satisfied with the outcome and reporting dabbing the eye with a tissue less than once per day, on a typical day (Munk score of 0 or 1).DCG was performed using a 24G Rabinov Sialography Set filled with Lipiodol Ultra fluid (dye). The lower canaliculus was cannulated to the midpoint. The tubing was taped to the cheek. Video fluoroscopy was performed as the doctor introduced the dye until the point the dye had reached the nasal space or significant reflux was observed. An erect x-ray was then taken 10 min later to determine if there was sequestration of dye within a closed cavity system.Data was analyzed using Microsoft Excel .Over a 4-year period, 160 eyes of 138 patients were identified  DCGs showed normal postoperative findings consistent with a successful DCR . Abnormal DCG findings were categorised into upper or lower canalicular block, common canalicular block, completely closed anastomosis, narrow anastomosis and anastomosis draining into a nasal sinus. Data for each of these categories are summarised in Table Of the 159 eyes, 94 (59%) underwent further corrective surgery  insertion was the most successful procedure in eyes with a normal DCG, resolving watering in 94% (17/18) of eyes. Redo-DCR was only effective at treating epiphora in 2/9 patient. Four patients in this redo-DCR failed group underwent tertiary LJT with success (4/4 resolution)., which resolved watering in 50% (1/2).Sisler trephination with tubes was the most successful procedure in eyes with an upper or lower canalicular blockRedo-DCR  with tubes was the most successful procedure in eyes with a common canalicular block with success in 83% (5/6).In eyes with completely closed anastomosis, LJT was successful in resolving epiphora in 1 eye (success rate of 100%). However, a much larger number of eyes  had redoRedo-DCR  was the most successful procedure and successfully resolved epiphora in 17 out of 27 eyes (success rate of 63%).Redo-DCR  was the only procedure performed in eyes whose anastomosis drained into a nasal sinus. This was performed in one patient with success (1/1).Patients were followed up for a median of 9.3 months (range 0.7–73.5) after their subsequent surgery.The surgical options for treating failed DCR surgery are well known, with surgeons tailoring their surgery to the approximate anatomical abnormality. In this study, we examined the DCG anatomical abnormality that led to DCR surgery failure, and reported the success rates of subsequent surgeries according to the anatomical abnormality.The most common DCG finding after failed DCR surgery was that of a well-sized and patent anastomosis with brisk flow of contrast into the nasal space (36%), which was followed by a narrow surgical anastomosis (31%), and completely closed anastomosis (19%). This study showed a similar percentage of DCG failure attributed to ‘inadequate ostium size or location’ 84/160, 53%) as that reported 30 years ago at this institution  3% as tha or a redThe results of subsequent surgery are presented in the flowchart, with the authors’ recommended surgical option for each DCG category circled in green Fig. . In all Mitomycin C (MMC) has been used with increased frequency as an adjuvant therapy in DCR surgery. While it has shown to increase surgical success in endonasal DCR revisions –15, thisRevision-DCR was successful overall in 61% (37/61) for all causes. However, when evaluated based on DCG findings of anatomical failure, rates of success ranged widely from 0 to 100%. When patients are weighing the risks vs. benefits of future surgery, the difference between a 0% or 100% predicted success rate becomes extremely important.Revision-DCR was least effective in patients with either upper or lower canalicular blockage (0% success) or a normal post-DCR DCG (22% symptom resolution). In patients with normal post-DCR DCG, this poor success rate is likely to reflect the fact that there may be other more proximal points of resistance to flow in the drainage system than at the nasolacrimal duct/anastomosis level. These patients typically have a high tear film, delayed fluorescein dye disappearance test and normal saline lacrimal irrigation test but delayed passage of fluorescein eye drops into the nasal space seen on endoscopy. These patients are described as having a poor lacrimal pump and likely have an unidentifiable  of the eyelid, punctum, canaliculus  or medial canthal tendon . In the Previous reports of symptomatic improvement after external redo-DCR range from 78%  to 85% , which pThree eyes (1.9%) of 3 patients had their lacrimal sac anastomosed to a nasal sinus rather than the nasal cavity. Unsurprisingly, all 3 patients had external approach DCR, and no tube stents were inserted in 2 cases. Nasal sinus anatomy is highly variable . In partMedicine is now entering into an era of shared decision making with the use of decision-making tools for patients. For this to be viable, patients need to be presented evidence for and against treatment options in a way that is easy for them to understand and compare. This flowchart Fig.  will serAlthough the cause of DCR failure can often be determined by careful clinical examination and nasal endoscopy, DCGs can identify precise structural abnormalities that would not be recognised through lacrimal irrigation alone Table . In thisAlthough DCGs carry radiation exposure, only 0.0011 to 0.0046Gy  is delivThe authors’ department benefits from access to walk-in Fluoroscopy for DCGs, facilitating same-day clinical assessment, DCG and discussion of results with the patient. However, we appreciate that this is not available in all clinical settings. Additionally, ordering a DCG means the cost of further appointment(s) as well as a delay in surgical planning. This may deter clinicians from using DCGs regularly. However, to a certain degree, surgeons and patients can still benefit from using this flowchart by substituting the DCG findings with clinical anatomical findings based on lacrimal syringing and nasal endoscopy Table .The small numbers in some of the categories make statistical comparisons limited and further studies with greater numbers will be needed to confirm these surgical success rates.The management of failure after DCR surgery represents a complex challenge. The structural abnormalities contributing to failure can be identified with DCG imaging. This is the first study to report success rates of redo-DCR surgery according to anatomical findings as confirmed by DCG. These results will help clinicians recommend procedures that are most likely to be successful for their patient’s specific anatomical abnormality, which can be determined by DCG or through a combination of lacrimal syringing and nasal endoscopy. The flow diagram is an easy to interpret visual tool for both physicians and patients, which can be used during a consultation, providing numerical evidence for the shared decision-making process.Additional file 1.


Mycobacterium tuberculosis (Mtb) is the causative agent of the infectious disease tuberculosis (TB), which is a leading cause of death worldwide. Approximately one fourth of the world's population is infected with Mtb. A major unresolved question is delineating the inducers of protective long-lasting immune response without inducing overt, lung inflammation. Previous studies have shown that the presence of inducible Bronchus-Associated Lymphoid Tissue (iBALT) correlate with protection from Mtb infection. In this study, we hypothesized that specific Mtb factors could influence the formation of iBALT, thus skewing the outcome of TB disease. We infected non-human primates (NHPs) with a transposon mutant library of Mtb, and identified specific Mtb mutants that were over-represented within iBALT-containing granulomas. A major pathway reflected in these mutants was Mtb cell wall lipid transport and metabolism. We mechanistically addressed the function of one such Mtb mutant lacking mycobacteria membrane protein large 7 (MmpL7), which transports phthiocerol dimycocerosate (PDIM) to the mycobacterial outer membrane (MOM). Accordingly, murine aerosol infection with the Mtb mutant Δmmpl7 correlated with increased iBALT-containing granulomas. Our studies showed that the Δmmpl7 mutant lacking PDIMs on the surface overexpressed diacyl trehaloses (DATs) in the cell wall, which altered the cytokine/chemokine production of epithelial and myeloid cells, thus leading to a dampened inflammatory response. Thus, this study describes an Mtb specific factor that participates in the induction of iBALT formation during TB by directly modulating cytokine and chemokine production in host cells. Mycobacterium tuberculosis (Mtb) is the causative agent of pulmonary tuberculosis (TB). TB is caused by inhalation of Mtb bacilli and it is currently estimated that approximately one fourth of the world's population is infected with Mtb , drophages , 4 (DCs)rophages –7 and harophages . We haverophages . During trophils , 11 and trophils , which h tissues . The earMtb and host specific factors that drive the formation of inducible bronchus associated lymphoid tissue (iBALT) during TB. To examine the Mtb specific factors involved, we utilized an Mtb transposon mutant library to screen the induction of lymphoid follicles using the Non-Human primate (NHP) model of pulmonary TB. This screen allowed us to identify Mtb genes which when mutated led to increased induction of iBALT within granulomas. The NHP model exhibits characteristics associated with human TB including the pulmonary cellular and acellular lesions  inner membrane protein is best known for the transport of phthiocerol dimycocerosate (PDIM) to the free fatty acid layer of the mycobacterial outer membrane (MOM) , a Mtb cell wall lipid, which can drive the observed decreased inflammatory cytokines and chemokine production by in vitro macrophages and also yields increased production of IL-10.In this study, we aimed to determine the  lesions , 14. We ne (MOM) , 16. Thene (MOM) –19, suggMtb exhibit the spectrum of disease severity observed clinically during human TB, with a diverse array of granuloma structures reflected by differences in immune cell recruitment and disease outcome  mutants from the himar1 transposon mutagenesis site hybridization (TraSH) library , were well-reflected. MmpL2 is a putative inner membrane transporter protein with unknown substrate, while MmpL7 is an inner membrane protein associated with phthiocerol dimycocerosate (PDIM) transport to the MOM  and lead Pb(II)   which ard Pb(II) .Mtb transposon screen conducted in NHPs has identified a novel set of Mtb genes involved in generation of lymphoid follciles within the lung, that appear to control Mtb metabolism and inner membrane lipid transport. In this study, we chose to mainly focus on the interaction driven by Mtb Mmpl7 that mediates formation of the granuloma.Taken together, the Mtb Mmpl7 in modulating iBALT generation in vivo, we infected C57BL/6 mice (B6) with 100 CFU Mtb Erdman or the Δmmpl7 mutant Erdman strain and characterized immune recruitment and induction of iBALT features in the lung. As previously shown, infection with the Δmmpl7 mutant resulted in early decreased Mtb CFU which recovered by 40 days post-infection (d.p.i.) in the lung and spleen  of Mtb Δmmpl7 to directly compare the ability of Δmmpl7 mutant to induce iBALT when compared to low dose (100 CFU) infection with wildtype (WT) Mtb Erdman  of the Δmmpl7 mutant and characterized myeloid and T cell accumulation in the lung. Upon infection with the Δmmpl7 mutant, we observed decreased neutrophils, monocytes and recruited macrophages at early time points as well as decreased accumulation of alveolar macrophages (AMs) and myeloid DCs (mDCs) when compared with accumulation in WT Mtb infected lungs , and IP-10 (CXCL10) that are associated with myeloid recruitment in ted mice . Interested mice . As IL-1ted mice , 31, theMtb Δmmpl7 mutant and decreased cytokine/chemokine production in vivo , in two human cohorts. We observed that G-CSF production was increased in ATB individuals when compared with LTBI or house contacts (HCs)  . Furtherts (HCs) , 34. As in vitro, which are a key source of G-CSF, and other inflammatory cytokines in the context of lung infection   when compared to WT Erdman   , using dl lipid) , 43. DATsurvival , 19. Thummpl7 mutant. To demonstrate this, we infected lung epithelial cells with Mtb Erdman and co-treated them with DATs. Our results show that co-treatment with DATs limited the production of G-CSF and IL-6 by WT Mtb infected epithelial cells  or the synthesis/translocation of virulence components including lipids & proteins .Although significant advances have been made in understanding the factors that aid in the intracellular survival of ClpB has already been established as essential for the in vitro growth of Mtb (Acr2 is yet to be established. Acr2 is the most up-regulated gene following phagocytosis of Mtb by macrophages (PhoP (Acr2 was strongly expressed in response to heat shock protein (HSP) Rv0251c and appears to play a role in early immune responses  mediated electron transfer to the electron transport chain  proteins and its disruption affects cell wall integrity leading to strong attenuation of the pathogen in a mouse model . Beyond reduced expression of PDIM, the Δmmpl7 mutant also has an established in vivo growth defect in the lung, spleen, and liver of mice, which we compensated for in our study by infecting with higher doses of the mutant compared to wild type. The decreased CFU in the lungs of Δmmpl7 mutant infected mice that we observed is similar to previous findings by Cox et al. and others , and not just stimulating heightened inflammatory responses by interactions with these receptors.Importantly, we observed increased IL-10 production in macrophages and DCs after Δoduction , suggestMmpL7 is colocalized with genes involved in polyketide biosynthesis (pks genes) and genes involved in lipid metabolism  suggesting that it is involved in complex lipid transport involving more than one substrate in Mtb . MmpL10 is considered to be the putative agent responsible for the transport of DAT across the plasma membrane  and the resultant downstream PRR signaling . Lipid vnfection . Moreovenfection . Hence tin vitro . This std Mincle .Mtb specific factors that drive these protective responses, and the mechanism of those interactions, are poorly understood. This study demonstrates that a single Mtb specific lipid factor can differentially affect the ability of various cell types to produce inflammatory and anti-inflammatory cytokines, all of which contribute in different aspects to the milieu that fosters conditions beneficial to iBALT formation. In the context of an immune structure as complex as iBALT, where multiple coordinated and regulated signals and cell types are required, future work will likely need to use a similar approach across several in vivo and in vitro model systems examining multiple cell types in order to elucidate the concise contribution of a specific Mtb factor and PRR interactions. Future work may also need to examine not only multiple cell types, but also locational and temporal cellular interactions throughout the course of infection, as a single Mtb factor is likely neither necessary nor sufficient in isolation to drive iBALT by interactions with a single cell type at one specific time point.While the various immune cells and cytokine/chemokine signals that contribute to iBALT formation are well-established in a variety of disease contexts , the MtbMtb genes and potential candidate lipids in skewing toward protective outcomes. We acknowledge that the role of any specific lipid in skewing iBALT formation is not an isolated phenomenon, as Mtb mutants knocking out certain lipids inevitably drive compensatory overexpression of others. Furthermore, we expect that the increased iBALT formation we have observed is likely due to a combination of a lack of PDIM along with increased DATs, with the possibility of other lipids being involved that we did not identify or assay for.While this study was not exhaustive in analyzing all potential lipid factors involved, it provides a framework for determining the contributions of the other Mtb cell wall lipids are critical to the initial interactions between Mtb and the host and suggest that Mtb specific lipids are key determinants of the early immune response that skews toward formation of iBALT. Our findings further provide a list of Mtb gene candidates for future work examining conditions needed for protective iBALT formation, specifically aimed at early responses that determine these outcomes. We have also demonstrated for the first time a panel of novel Mtb genes that are associated with enhanced formation of protective iBALT containing granulomas and improved disease outcomes, while most work on this topic examines genes involved in detrimental host outcomes and loss of protection. This study thus provides a framework for future attenuated vaccine candidates and mechanistic studies across model systems.Our novel findings demonstrate that genes associated with Mtb (H37Rv) transposon site hybridization mutants (TraSH) library (kind gift of Dr. Chris Sassetti) was delivered high dose  CFU via bronchoscopic procedure into the lungs of triplicate Indian rhesus macaques. Delivery of bacteria to the lower lungs was confirmed via dilution plating and Mtb infection was confirmed by direct observation of the clinical signs of TB and concurrent positive tuberculin skin test. NHPs were humanely euthanized after being held 4–6 weeks due to development of disease using prespecified criteria  mice used were 6–8 weeks old and sex matched. All treatments and conditions used to handle study animals are in accordance with the approved Institutional Animal Care and Use Committee (IACUC) guidelines at Washington University in St. Louis.Mtb (Erdman) and ΔmmpL7 mutant (MJM39) (Mtb in the above Glasscol nebulizer at tested doses of ~100 CFU (low) or ~500 CFU (5-fold higher) of Mtb per mouse. At the indicated time points mice were sacrificed via carbon dioxide narcosis and lungs and spleens were harvested. (MJM39)  were stoLung pathology from formalin fixed paraffin embedded lungs was assessed digitally using the automated tool of the Zeiss Axioplan 2 microscope (Carl Zeiss) of H&E stained slides to quantify inflammation and immunofluorescence labeled slides B220, CD3 and counter stained with DAPI in order to assess lymphoid follicle area.Bacterial burden was assessed using serial 10-fold dilutions of lung or spleen and plated on 7H11 supplemented with OADC . After 2–3 weeks incubation colonies were counted visually. Cytokine/chemokine expression was analyzed in lung homogenates from infected mice via Luminex (Millipore-Sigma) or ELISA (R&D).Flow cytometry was conducted on single cell preparations derived from infected and uninfected lungs using fluorochrome conjugated antibodies:−, CD11c+, Siglec-F+, CD64+. mDCs: CD11b+CD11c+, MHC-II+. Neutrophils: CD11b+, CD11c−, Gr-1hi. Monocytes: CD11b+ CD11c− Gr-1int and/or Ly6C+. Recruited macrophages: CD11b+ CD11c−, Gr-1lo, CD64+/−, Ly6C+/−, MHC-II+/−.Myeloid antigen presenting cell panel : CD11b , apoptotic , and dead .Live, apoptotic, or dead cell panel : AnnexinData collection and analysis were conducted on the BD LSR Fortessa, Fortessa-X20 Cytometers or the FACSJazz Cell Sorter, all with FACS Diva software and post-acquisition analysis conducted on FlowJo.Mtb. Fresh blood samples from active pulmonary TB patients were obtained from patients recruited to the Tuberculosis Outpatient Clinic, INER, Mexico, or the KwaZulu-Natal Research Institute for TB and HIV, South Africa. Serum samples from all patients with active TB were collected prior to anti-Mtb treatment and did not presented comorbidities such as diabetes, HIV, cancer, and COPD. LTBI patients were defined as asymptomatic individuals who had positive IGRA test results as previously described , KwaZulu-Natal Research Institute for TB and HIV (Currently renamed AHRI), South Africa and The American British Cowdray Medical Center. All subjects were of similar socioeconomic status and unrelated to the third generation as determined by a questionnaire. TB cases had symptoms  and chest radiographic findings consistent with recent pulmonary TB, a positive sputum acid-fast smear and culture confirmed for in vitro infection hemocytometer counts from a representative well were used to calculate multiplicity of infection (MOI). C10 cells were washed once with sterile PBS and then infected with Mtb or Mtb mutant at an MOI of 1 in DMEM without antibiotics and incubated at 37°C, 7.5% CO2. Supernatants from the infection was collected and stored at −80°C. Cells were collected by tryspinization and stained as per manufacturer's protocol.Mouse lung epithelial cell line C10 cells were cultured to confluence in 24 well plates in DMEM. Prior to 6 cells per ml and supplemented with 4% GM-CSF for 7 days. At harvest, adherent cells were collected as BMDMs, and floating cells were collected as BMDCs, gently washed with CDMEM via centrifugation , resuspended and then plated at 1 × 106 cells per ml.Primary B6 mouse bone marrow derived macrophages (BMDMs) and dendritic cells (BMDC) were prepared as previously described . Briefly9 Polybead polystyrene beads) were washed twice in 0.05 M carbonate-bicarbonate buffer (pH 9.6) and then incubated with 50 μg of DATs or buffer alone for 1 h at 37°C. Beads were then blocked with 5% BSA, washed repeatedly in 0.5% BSA, and finally adjusted to 4.0 × 108/ml in 0.5% BSA before being used  were extracted with chloroform:methanol  following the previously published method . DATs were purified by preparative TLC as previously described for other Mtb cell envelope lipids  using unpaired two tailed Student's All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by AHRI and INER ethics board. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Washington University in St. Louis, Tulane Primate Center.MD and OP designed and performed experiments, analyzed data, and prepared the manuscript. KT, JR-M, JS, and JT performed experiments, provided samples, and generated data. JC, JZ, and AS provided samples or reagents. DK and SK designed experiments and provided guidance and funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our results demonstrate that the new high-density exon-focused targeted array design that uses updated information from large scale genomic studies is a powerful tool for detection of intragenic deletions and duplications that leads to a significant improvement in diagnostic yield.In recent years, chromosomal microarrays have been widely adopted by clinical diagnostic laboratories for postnatal constitutional genome analysis and have been recommended as the first-line test for patients with intellectual disability, developmental delay, autism and/or congenital abnormalities. Traditionally, array platforms have been designed with probes evenly spaced throughout the genome and increased probe density in regions associated with specific disorders with a resolution at the level of whole genes or multiple exons. However, this level of resolution often cannot detect pathogenic intragenic deletions or duplications, which represent a significant disease-causing mechanism. Therefore, new high-resolution oligonucleotide comparative genomic hybridisation arrays (oligo-array CGH) have been developed with probes targeting single exons of disease relevant genes. Here we present a retrospective study on 27,756 patient samples from a consortium of state-funded diagnostic UK genomic centres assayed by either oligo-array CGH of a traditional design (Cytosure ISCA v2) or by an oligo-array CGH with enhanced exon-level coverage of genes associated with developmental disorders . The new targeted design used in Cytosure v3 array has been designed to capture intragenic aberrations that would have been missed on the v2 array. To assess the relative performance of the two array designs, data on a subset of samples ( G-band karyotyping, the previously recommended test for patients with such phenotypes, provides a diagnostic yield of approximately 3%, while chromosomal microarray (CMA)-based testing has been shown to offer yields of 10–20%2.Over the last decade, the implementation of comparative genomic hybridisation arrays (array CGH) as a first-tier test for detecting chromosomal aberrations associated with developmental delay (DD), intellectual disability (ID), autism spectrum disorder, multiple congenital anomalies and other neurodevelopmental phenotypes has shown a marked improvement in diagnostic yields4) and the cumulative knowledge gained through the sharing of high-resolution, highly-curated genomic data in easily accessible public databases . A number of research groups have also advocated the use of exon-level probe coverage for specific genes, allowing further increases in diagnostic yield through the detection of smaller intragenic copy number variants (CNVs)6.Modern oligonucleotide-based array designs have typically focused on providing broad genome coverage with higher probe density in regions associated with specific disorders. More recently, newer array designs have been introduced that incorporate the findings of large-scale genomic studies  for diagnostic testing of National Health Service (NHS) patients. The previous version, the CytoSure International Standards for Cytogenomic Arrays (ISCA) v2 (hereafter referred to as CytoSure v2), focuses on known disease-causing genes and has a “backbone” of probes across the entire genome. The new CytoSure v3 is a more focused, exon-centric oligo-array design that incorporates novel content from the DDD study and ClinVar database. Similar to the CytoSure v2 design, the v3 array has a reduced coverage of probes across uninformative regions to avoid detection of variants of unknown significance and incidental findings. We compare the performance of the CytoSure v3 array with that of the CytoSure v2 array; and also interrogate the entire post-natal proband dataset, providing further information on the type , size, frequency and chromosomal distribution of CNVs across the population tested.We analysed anonymised data from 27,756 postnatal blood samples obtained from four NHS laboratories. In total, 95,273 CNVs were identified, equating to an average of 3.43 CNVs per sample , likely pathogenic or pathogenic are reported, a reporting rate was calculated for all samples Table , which eThis dataset includes two laboratories that transitioned from the v2 oligo-array to the v3 after in-house verification, and two laboratories that have only used the v3 array. In order to reduce the impact of inter-laboratory variation, only data from the two laboratories that ran both arrays were included in these analyses. In total, 19,675 samples were analysed, comprising 16,830 samples (85.54%) run on the CytoSure v2 array and 2845 samples (14.46%) run on the CytoSure v3 array.When comparing sample classifications for both oligo-array designs, the largest difference is the higher proportion of samples classified as likely pathogenic using the CytoSure v3 array  for the CytoSure v3 array . The proportion of samples classified as VOUS is comparable (~10%) for both oligo-array designs. Comparing the reporting rate between the two array designs shows a significant 4.49% increase  or by an oligo-array CGH with enhanced exon-level coverage of genes identified as being important in developmental disorders from research projects (Cytosure v3). In total, 95,273 CNV calls were identified with an average of 3.43 CNVs per sample. Pathogenic or likely Pathogenic CNVs were found in 13.43% of all patient samples. When comparing the two array designs, an additional 4.72% of samples were found to have a likely pathogenic or pathogenic call when using the CytoSure v3 array in comparison to the CytoSure v2 array. Overall, the diagnostic yield in our cohort was comparable with previous studies in patients with ID and DD, where the detection rate of pathogenic array CGH identified by array CGH ranged from 7 to 20%P = 2.85e–07) compared to the CytoSure v2 design. This is due to an increase detection of clinically significant CNVs classified as pathogenic or likely pathogenic while mitigating an increase in VOUS rate, indicating a well-balanced design of the new v3 array.The rate of reportable CNVs  reached 22.72% with more than one reportable CNV identified in 5.07% of the reportable cases. As expected, the CytoSure v3 array showed a significant improvement in reporting rate  of intragenic CNVs and was able to call small likely pathogenic and pathogenic intragenic aberrations in known ID genes that would have been missed on the CytoSure v2 array  has recently issued new guidelines for the interpretation and reporting of CNVs in routine diagnostics25. Targeted arrays offer a stable, proven platform with simple data analysis and minimal demands on bioinformatics capacity, a common limiting factor for some laboratories when adopting NGS as a routine test to detect CNVs in a large cohort of patients. To ensure the continued targeting of the most disease-relevant regions, CMA design must be routinely updated to incorporate latest genomic information delivered by high-resolution analysis technologies and the findings of large genomic projects.Until NGS-based approaches progress to be able to reliably detect CNVs in a routine diagnostic setting, exon-focused CMAs will remain an important complementary tool to WES and WGS for analysis of both large and small genomic copy number imbalancesIn conclusion, we report a measurable improvement in diagnostic yield in NHS patients assayed by an oligonucleotide array CGH with enhanced exon-level coverage of genes important for developmental disorder research (Cytosure v3) in a very large cohort from a consortium of state-funded diagnostic genomic centres in the UK. Our results also show that the new exon-focused array design provides a powerful tool for detection of small pathogenic intragenic deletions and duplications, providing a clinical answer for a proportion of patients that would have remained undiagnosed. At this time, targeted arrays offer a robust method of copy number analysis to supplement NGS based tests.Developing a curated dataset suitable for analysis required array processing, calling of CNVs, data acquisition, classification binning and data filtering. All samples and data were collected retrospectively from a cohort of patients with neurodevelopmental disorders who underwent routine phenotypic-driven diagnostic testing in the NHS setting. Since the aim of this study is to assess the performance of the CytoSure v3 array platform across multiple ISO 15189 UKAS accredited diagnostic genetic laboratories in a routine NHS diagnostic setting, the acquired data has been anonymised and contained no further clinical information  in order to maintain patient confidentiality. All contributing laboratories performed their own technical verifications, including calculating performance parameters and running samples in parallel, to satisfy ISO 15189 requirements. For example summary of validation data see Supplementary Note Clinical consent was obtained for all samples prior to genetic testing. For our retrospective analysis, we used anonymised data from existing diagnostic samples for which formal ethical approval was not required. Our study did not involve prospective collection of samples or changing care from accepted standards for any of the patients. The study was approved by individual Trusts as a service evaluation and an approval from Information Governance in each Health Board was obtained to collect the anonymised data.DNA were extracted from post-natal patient samples using laboratory-specific protocols. DNA samples were labelled using the CytoSure Genomic DNA Labelling Kit prior to running on the CytoSure ISCA v2 (8 × 60 k) array or CytoSure Constitutional v3 (8 × 60 k) array according to the manufacturer’s  guidelines. Aberration detection was performed using CytoSure Interpret Software according to analysis protocols validated by each contributing laboratory.CNV detection within CytoSure Interpret Software is carried out using a circular binary segmentation algorithm in order to group probes into regions of equal copy number. Thresholds are applied based on log ratio and region size to determine which segments are significant and therefore represent a loss or a gain. These segments are hereby described as calls.Each contributing laboratory deploys their own call classification structures. To allow consistency of data comparison, all participating laboratories were asked to ‘bin’ their classification structure into five standardised classifications , which were subsequently used for all downstream analyses.After filtering , only data from the two laboratories that had utilised both array designs were used in these analyses.All statistical analyses were performed in R version v3.5.0. The Test of Equal or Given Proportions was used for all comparisons.The reporting rate was calculated based on the assumption that only samples with one or more CNV classified as VOUS, likely pathogenic or pathogenic are reported [Eq. ]. AlthouFurther information on experimental design is available in the Supplementary InformationReporting SummarySupplementary Data 1