Carbohydrate response element binding protein (ChREBP) is a transcription factor that responds to glucose and activates genes involved in the glycolytic and lipogenic pathways. Recent studies have linked adipose ChREBP to insulin sensitivity in mice. However, while ChREBP is most highly expressed in the liver, the effect of hepatic ChREBP on insulin sensitivity remains unknown. To clarify the importance of hepatic ChREBP on glucose homeostasis, we have generated a knockout mouse model that lacks this protein specifically in the liver (Liver-ChREBP KO).Using Liver-ChREBP KO mice, we investigated whether hepatic ChREBP deletion influences insulin sensitivity, glucose homeostasis and the development of hepatic steatosis utilizing various dietary stressors. Furthermore, we determined gene expression changes in response to fasted and fed states in liver, white, and brown adipose tissues.Liver-ChREBP KO mice had impaired insulin sensitivity as indicated by reduced glucose infusion to maintain euglycemia during hyperinsulinemic-euglycemic clamps on both chow (25% lower) and high-fat diet (33% lower) (p < 0.05). This corresponded with attenuated suppression of hepatic glucose production. Although Liver-ChREBP KO mice were protected against carbohydrate-induced hepatic steatosis, they displayed worsened glucose tolerance. Liver-ChREBP KO mice did not show the expected gene expression changes in liver in response to fasted and fed states. Interestingly, hepatic ChREBP deletion also resulted in gene expression changes in white and brown adipose tissues, suggesting inter-tissue communication. This included an almost complete abolition of BAT ChREBPβ induction in the fed state (0.15-fold) (p = 0.015) along with reduced lipogenic genes. In contrast, WAT showed inappropriate increases in lipogenic genes in the fasted state along with increased PEPCK1 in both fasted (3.4-fold) and fed (5.1-fold) states (p < 0.0001).Overall, hepatic ChREBP is protective in regards to hepatic insulin sensitivity and whole body glucose homeostasis. Hepatic ChREBP action can influence other peripheral tissues and is likely essential in coordinating the body's response to different feeding states. •Liver ChREBP is protective in maintaining hepatic insulin sensitivity and regulating hepatic glucose production.•Hepatic ChREBP deletion protects against carbohydrate-diet induced hepatic steatosis but this is not metabolically favorable.•Deletion of hepatic ChREBP alters gene expression in adipose tissues suggesting complex inter-tissue communication. When food is abundant, energy must be stored for later use, and, during fasting, it must be mobilized and delivered throughout the body. Key tissues involved in these processes are the liver, muscle and adipose tissues. The reliance of various tissues in the body on glucose as a fuel substrate means blood glucose concentration in particular must be maintained. This means during periods of fasting the liver releases glucose into the bloodstream. One of the key actions of insulin in the fed state is to suppress this hepatic glucose production as well as increase uptake of glucose in muscle and adipose tissues. Impairments in the action of insulin can lead to impaired fasting glucose or glucose intolerance which are key risk factors for the development of diabetes de novo lipogenesis, the association found with hepatic steatosis is unsurprising. Selective over-expression of ChREBP in the liver of mice worsens hepatic steatosis A key transcription factor involved in coordinating the feeding response is carbohydrate response element binding protein (ChREBP). ChREBP is a transcription factor that regulates numerous genes in response to changes in cellular glucose concentration. Pathway analysis of ChREBP target genes has revealed roles for ChREBP in lipid and carbohydrate metabolism, cell motility, and insulin signaling We sought to clarify the role of hepatic ChREBP in glucose homeostasis and insulin sensitivity using a novel mouse model of hepatic ChREBP deletion. We describe the physiological consequences of specific liver ChREBP deletion in mice and have identified an important role for hepatic ChREBP in whole body glucose homeostasis and hepatic insulin sensitivity.22.1Liver-specific ChREBP KO mice were created by breeding floxed mice  to a line expressing the Cre recombinase specifically in liver (Alb-cre). The resultant homozygous progeny (Liver-ChREBP KO) do not show expression of ChREBP in the liver. Group housed animals were kept on a 12-h light–dark cycle with free access to food and water, with body weights recorded weekly. All conditions and experiments were reviewed and approved by Monash University Animal Ethics committee. Experimental diets were generated by Specialty Feeds . High fat diet  was based on Research Diets D12451. High carbohydrate diet  was created using SF04-001 as a template with similar vitamin and mineral composition. Detailed comparison of the high-fat and high-carbohydrate diets is given in 2.2Body composition was assessed by dual-energy X-ray absorptiometry (DEXA) .2.3Mice were individually housed in metabolic chambers  in order to assess metabolic activity. Mice were acclimated for 48 h before data were collected. Mice had free access to food and water for baseline recordings, or were subjected to a fasting-refeeding protocol in which food was removed for 24 h at the beginning of the night period, and recordings were taken during fasting, refeeding, and recovery. Oxygen consumption, carbon dioxide production, and ambulatory activity were measured, and RER and energy expenditure were calculated.2.4To assess glucose tolerance, 18-hour fasted animals were dosed with a 3 g/kg glucose solution P.O. and blood glucose was measured at 0, 30, 60, and 120 min via tail blood using a handheld glucometer .2.5Mice were subjected to jugular vein catheterization 5 days prior to hyperinsulinemic euglycemic clamp studies. Mice were anesthetized with isoflurane (2–3% in oxygen) while an indwelling silastic catheter was inserted into the right internal jugular vein and exteriorized through the back of the neck. The catheters were kept patent with heparin sodium  and sealed with a stainless steel plug. Mice were allowed 4–5 days postsurgical recovery. Food pellets were placed at the bottom of the cage to facilitate recovery. Body weight was recorded daily, and mice that had less than 5% weight loss were subsequently studied.Hyperinsulinemic euglycemic clamps followed by 2-deoxyglucose uptake were performed on 6 h fasted, conscious, and unrestrained mice as described previously 2.6For lipid staining, livers were frozen in liquid nitrogen-cooled isopentane then embedded in OCT . Frozen blocks were sectioned at 10 μm, mounted and dried, and stained with Oil Red O or Hematoxylin & Eosin. For trichrome staining and periodic acid-Schiff (PAS) staining, livers were preserved in 10% formalin (w/v) for 24–48 h followed by embedding in paraffin, sectioning at 4 μm and staining with Masson's trichrome, PAS, or Hematoxylin & Eosin.2.7To measure liver glycogen, 200 mg frozen liver was homogenized in 20 mM Tris HCL with 0.1% Triton-X100. Samples were spun at 2700 × g at 4 °C for 20 min, and the supernatant was used for protein BCA assay and glycogen assay. For glycogen assay, samples were precipitated overnight at 4 °C with ethanol, reconstituted with 0.04 M Na acetate buffer followed by incubation in Na acetate buffer either with or without amyloglucosidase for 1 h at 37 °C. The hydrolyzed free glucose concentration was measured with Glucose (HK) Assay Kit .2.8For all experiments, a modified Kreb's-Henseleit buffer was gassed for 40 min with 95% O2/5% CO2. Glucose (5 mM) and fatty acid-free BSA (4%) was added to the buffer immediately before experiments. All experiments were conducted in a shaking water bath at 30 °C.For hepatic lipogenesis d-[3-3H]Glucose  was added to the buffer to give a final concentration of 0.5 μCi/ml. Liver was sliced into 1–2 mm explants and incubated for 2 h, and the medium was removed. The tissue was washed in PBS and then homogenized in 1 ml PBS. The lipids were extracted in 2:1 chloroform-methanol, a 1-ml aliquot of the organic phase was removed, scintillation fluid was added, and radioactivity was counted in a liquid scintillation analyzer.−1 of [1-14C]palmitate .For analysis of oxidation all experiments, liver was sliced into 1–2 mm and explants were placed in warmed (30 °C) Krebs-Henseleit buffer pH 7.4 containing 2 mm pyruvate, 4% fatty acid-free bovine serum albumin  and 1 mm palmitic acid . After an initial incubation of 20 min, the incubation buffer was replaced with the same buffer described above supplemented with 0.5 μCi ml2.9Blood samples were obtained by retro-orbital bleeds under isoflurane anesthesia. Plasma glucagon, ghrelin, GIP, GLP-1, insulin, leptin, PAI-1, and resistin were determined by multiplexed bead assay  using the MAGPIX instrument (Luminex).Serum triglyceride and fatty acid levels were determined by colorimetric reaction .2.10RNA was extracted from liquid nitrogen flash-frozen samples and purified using a column-based RNeasy 96 QIAcube HT Kit . Extracted RNA was quantified and checked for purity using the QIAxpert system (Qiagen). cDNA was generated from 1 μg RNA using iScript Advanced cDNA Synthesis Kit for RT-qPCR . qPCR was run for ChREBP-α , ChREBP-β  and 36B4  (reference gene) using custom oligonucleotides  and SYBR Green PCR Master Mix . Amplifications were performed using an Applied Biosystems 7900HT instrument, followed by a melt curve analysis.Additionally, qPCR was performed for other target genes using Taqman assays  and the 2.11Stata Statistical Software: Release 14, College Station, TX; StataCorp LP). Additional graphs and statistical analyses were completed using GraphPad Prism  as indicated.All data are presented as mean ± SEM, with a statistically significant difference defined as p < 0.05. For the analysis of calorimetry data, general estimating equation models were used to provide population average effects to take into account the correlation across time. Adjustment was undertaken to assess the effects of individual total body weight, lean and fat mass. All graphs and statistical analyses were completed using StataCorp 2015 software  was performed. Liver-ChREBP KO mice showed elevated blood glucose 30 min post receiving a glucose load orally, suggesting an impairment in the ability to clear glucose A p < 0.B p < 0.C,D p < E,F. In s3.2ChREBP is a lipogenic transcription factor that is known to be involved in the development of hepatic steatosis. In order to determine the effect of hepatic ChREBP deletion on hepatic steatosis development, livers were histologically assessed for fat deposition via Oil Red O staining in Liver-ChREBP KO and control mice fed either a normal chow, a high-fat diet or a high-calorie high-carbohydrate diet. A representative image from Liver-ChREBP KO and wildtype or Fl/Fl control mice fed a chow, high-fat or high-carbohydrate diet for 12 weeks is displayed in 3.3In order to assess the effect of hepatic ChREBP deletion on liver metabolic function, glycogen content, fatty acid oxidation, and lipogenesis were measured in chow-fed mice. There was no significant difference in liver glycogen content between genotypes, suggesting no impairments with glycogen synthesis and storage in Liver-ChREBP KO mice A. This w3.4Liver-ChREBP KO mice were confirmed to have a selective ChREBP deletion in liver, with expression still evident in other tissues . As tiss3.5ChREBP is a transcription factor that regulates various genes involved in glycolysis, lipogenesis, and glucose metabolism. In order to look at the effect of hepatic ChREBP deletion on the mRNA expression of these genes, Liver-ChREBP KO and control mice were either fasted overnight or left in the fed state, and various tissues were collected for gene expression analysis. In addition to ChREBP target genes, other lipogenic transcription factors were measured to assess any potential compensation occurring due to hepatic ChREBP deletion. Furthermore, various genes can regulate ChREBP or are involved in feedback pathways with ChREBP, and these genes were also measured. Finally, as Liver-ChREBP KO mice seem to be in a heightened inflammatory state, as demonstrated by their increased plasma resistin and PAI-1, expression of various inflammatory markers was also measured.Liver-ChREBP KO mice had various gene expression changes in the liver. In the fasted state, Liver-ChREBP KO mice had elevated liver type pyruvate kinase (L-PK) (2.3-fold) (p < 0.0001) as well a trend towards increased acetyl-coA carboxylase 1 (ACC1) (1.5-fold) and fatty acid synthase (FAS) (1.6-fold) A. In theThe gene expression changes in WAT show inappropriate upregulation of lipogenic genes in the fasting state in Liver-ChREBP KO mice. Expression of ACC1 (2.7-fold), SCD1 (2.0-fold) and thioredoxin interacting protein (TXNIP) (2.1-fold) are all increased in fasted Liver-ChREBP KO mice when compared to control mice A p < 0.. InteresThe gene expression changes seen in BAT were greatly different to those seen in WAT. In the fasted state, there was a trend towards a decrease in the ChREBP target genes ACC1, FAS, SCD1, and FGF21 in Liver-ChREBP KO mice (0.3-fold) A. This r4In this study, we characterized a novel mouse model of hepatic ChREBP deletion to investigate the importance of this factor in glucose homeostasis and insulin sensitivity. We found liver ChREBP is essential for maintaining whole-body glucose homeostasis and hepatic insulin sensitivity. Liver-ChREBP KO mice had reduced fat mass but gained similar weight when fed a high-fat diet. Regardless of diet, these mice displayed glucose intolerance and insulin resistance with specific impairments in the suppression of hepatic glucose production. Along with the reduced liver insulin sensitivity, hepatic ChREBP deletion impaired the lipogenic transcriptional response to fasting and feeding suggesting a lack of appropriate compensation by alternate lipogenic transcription factors to glucose sensing. Apart from these effects on the liver, there were also dramatic gene expression changes evident in both white and brown adipose tissues. Finally, although ChREBP deletion did protect against carbohydrate-diet induced hepatic steatosis this was not protective in terms of metabolic health. The alterations in liver in response to ChREBP deletion have wide-ranging effects on whole body glucose homeostasis and can induce specific changes in WAT and BAT to affect lipid homeostasis.Perhaps the most striking effect of hepatic ChREBP deletion was the impairment of hepatic insulin sensitivity. Liver-ChREBP KO mice displayed glucose intolerance, which was most likely due to insulin no longer being able to suppress hepatic glucose production (HGP) effectively. Importantly, this impairment was also seen after high-fat diet feeding, despite no differences in body composition. This suggests that ChREBP action in the liver is important in maintaining hepatic insulin sensitivity. This contradicts human association studies showing an association of insulin resistance with increased liver ChREBP Deletion of hepatic ChREBP may also affect indirect mechanisms of HGP regulation. It is known that insulin can directly suppress HGP through hepatic Akt signaling resulting in inhibition of FOXO1. Yet, studies have shown insulin acts through direct and indirect pathways in humans to suppress HGP de novo lipogenesis in brown adipose tissue increases supply of fatty acid substrates. In fact, although acute activation of β3-adrenoceptors, which mediate sympathetic activation of thermogenesis, suppresses de novo lipogenic genes chronic administration increases them Hepatic ChREBP deletion resulted in profound alterations in brown adipose tissue gene expression. Strikingly, the induction of BAT ChREBPβ expression in the fed state was almost completely abolished in Liver-ChREBP KO mice. This was accompanied by a trend towards decreased lipogenic gene expression in both fed and fasted states. The role of ChREBP in BAT is not completely understood, but it has been shown to induce lipogenesis and is involved in a feedback loop with PPARα to coordinate appropriate lipogenesis and lipolysis de novo lipogenesis may be beneficial Although hepatic ChREBP deletion can protect mice from carbohydrate-diet induced hepatic steatosis this is not protective in terms of metabolic health. Despite reductions in liver triglyceride, Liver-ChREBP KO mice fed a high-carbohydrate diet display worsened glucose tolerance and increased serum PAI-1 and resistin . This sude novo lipogenesis and fatty acid oxidation in WAT By examining the effects of hepatic ChREBP deletion, a complex inter-tissue communication network to maintain metabolic homeostasis can be observed. Deletion of ChREBP in liver impairs the normal response to fasting and feeding as observed by the lipogenic gene expression profiles. Furthermore, Liver-ChREBP KO mice have reduced hepatic fatty acid oxidation, suggesting alterations in substrate utilization. This is seen at a whole body level with changes in the respiratory exchange ratio reflecting increased oxidation of carbohydrates, particularly after fasting. The liver is the central organ of glucose and lipid metabolism, so it is unsurprising that alterations here would impact other metabolic tissues. Although adipose tissue and adipokines have recently garnered the most attention in their ability to influence whole body insulin and glucose homeostasis, the liver undoubtedly plays a role as well. In fact, this has been shown previously where lipids from the liver can regulate In conclusion, we have characterized a novel mouse model to examine the importance of hepatic ChREBP on whole body glucose homeostasis and insulin sensitivity. Liver ChREBP is vital in maintaining hepatic insulin sensitivity and coordinating the appropriate responses to fasting and feeding through glucose sensing. Future investigations to identify the mediators of these responses may reveal novel pathways that could be exploited for therapeutic development for hyperglycemia and diabetes.


Tracheobronchial anomalies are common in congenital heart disease (CHD), including tracheobronchial stenosis, tracheal bronchus, cardiac bronchus, and bronchial isomerism, which can cause varying degrees of respiratory illness. It is necessary to assess tracheobronchial anomalies and make a preoperative airway evaluation. Multi-slice computed tomography (MSCT) and cardiac magnetic resonance imaging (MRI) are the most effective noninvasive modalities for the diagnosis of CHD and the associated tracheobronchial anomalies. However, MSCT remains an ionizing procedure despite using low dose protocols. The aim of this study was to evaluate diagnostic accuracy of tracheobronchial anomalies in patients with CHD using three-dimensional turbo field echo(3D-TFE) magnetic resonance imaging sequence for preoperative airway evaluation. The results indicated that 3D-TFE provided better image quality as compared to that of 3D-balanced turbo field echo (3D-bTFE), and it can clearly demonstrated the tracheobronchial tree and tracheobronchial anomalies in CHD. This study confirms the clinical value of 3D-TFE in diagnosing tracheobronchial anomalies and supply helpful tracheobronchial information for preoperative strategies and postoperative follow-up. Tracheobronchial stenosis can cause severe respiratory illness. In our institution, the cardiothoracic surgery service routinely requires imaging for airway anatomy prior to surgery. MSCT and cardiac MRI are the most effective noninvasive modalities for the diagnosis of CHD and the associated tracheobronchial anomalies8. However, MSCT remains an ionizing procedure despite using low dose protocols4. MRI has the advantage of being non-ionizing and providing excellent soft tissue contrast for the diagnosis of CHD and tracheobronchial anomalies. Spin echo (SE) sequence can demonstrate the tracheobronchial tree6, but it is typically a 2D sequence and therefore difficult to depict the entire tracheobronchial tree optimally. 3D sequences which include 3D-bTFE8 and 3D-TFE can delineate the entire tracheobronchial tree clearly through post-processing. The aim of this study was to evaluate diagnostic accuracy of tracheobronchial anomalies in patients with CHD using 3D-TFE magnetic resonance imaging sequence for preoperative airway evaluation.Tracheobronchial anomalies including tracheobronchial stenosis, tracheal bronchus, cardiac bronchus, and bronchial isomerism are common in congenital heart disease. Cardiovascular anomaly is the principal extrinsic lesion causing tracheobronchial stenosis, including vascular rings, left pulmonary artery sling, and absent pulmonary valve, etcIn 75 patients, 25 patients were diagnosed cyanotic CHD and 50 patients were acyanotic CHD. . The difference of the image quality scores between 3D-TFE and 3D-bTFE by the two reviewers were statistically significant (p < 0.01).Excellent image quality scores of 3D-TFE (4.61 ± 0.44 and 4.61 ± 0.42) from reviewer 1 and 2 respectively were exhibited in 71 patients. The image quality scores of 3D-bTFE were (3.93 ± 0.54 and 3.95 ± 0.55) and the agreement between the two reviewers was good  and can demonstrate the tracheobronchial tree but the margin of upper portion trachea was not well visualized due to loss of signal compared with 3D-TFE Fig. . There w4. CHD is often associated with tracheobronchial anomalies4. In our center, the patients who will have corrective surgery for CHD will undergo MSCT or cardiac MRI for preoperative airway evaluation.Airway disorders are potentially life-threatening, and the interpretation of chest radiographs or images from any other chest imaging modality in the pediatric population is incomplete without airway evaluation9. MSCT plays an important role in noninvasive imaging of the lungs and airway in pediatric patients due to its higher spatial resolution, greater speed and lower need for sedation12. However, MSCT utilizes ionizing radiation even using low dose protocols, and vascular studies require intravenous injection of iodinated contrast agents13.MSCT and cardiac MRI are the most effective noninvasive modalities for the diagnosis of CHD with tracheobronchial anomalies8. The signal void of blood flow in SE sequences provides a good contrast between the vascular lumen and the vessel walls and excellent differentiation between surrounding soft tissues and mediastinal fat, providing additional diagnostic information. In the 1990s, the T1 weighted (T1W) SE sequence was the principal tool for the demonstration of tracheobronchial anomalies16. However, T1W SE with thin slices requires a long acquisition time and is sensitive to motion and 2D images produce suboptimal 3D multi-planar reconstructions; Hence, the three-dimensional anatomy of the tracheobronchial tree cannot be demonstrated adequately. In recent years, authors have noted that varying degrees of airway narrowing can be noted on 3D-bTFE images19. Then, by utilizing 3D-TFE, we detected 64% tracheobronchial anomalies in our group . 3D-TFE provided improved image quality as compared to that of 3D-bTFE. All patients underwent MSCT before or after MRI examination and there was a high inter-modality agreement between MSCT and 3D-TFE (Kappa = 0.75). The sensitivity, specificity and accuracy of 3D-TFE were higher than 3D-bTFE. The higher NPV and lower NLR of 3D-TFE indicate that 3D-TFE sequence can demonstrate normal tracheobronchial pattern more correct than 3D-bTFE sequence. Although the PPV of 3D-TFE(89.0%) was lower than 3D-bTFE(91.3%), the difference between them is not obvious.MRI is widely used in anatomy and function assessment of CHD due to the absence of ionizing radiation19. Navigator gating and ECG triggering are enabled to reduce artifacts from respiratory and cardiac motion. In MinIP reconstruction, the tracheobronchial tree can be visualized optimally. Performing 3D-TFE after contrast injection also enhances the contrast between the tracheobronchial tree and surrounding tissues. The relationship between the tracheobronchus and its surrounding vascular structures can be demonstrated through MIP reconstruction. 3D images overcome the disadvantages of axial images, such as the inability to identify focal narrowing, reliably estimate the longitudinal extent of disease and display complex 3D anatomic relationships.3D-TFE is a 3D fast gradient echo sequence that employs an inversion recovery radiofrequency pulse for magnetization preparation. It is designed for rapid acquisition with T1 weighting dominance. Fast gradient echoes are characterized by their rapid sampling time and high signal intensity and image contrast when approaching steady state. The rapid speed of acquisition makes 3D-TFE an excellent alternative cardiac imaging modality. Without fat suppression and T2 prep pulses in 3D-TFE sequence makes further enhance the contrast between the tracheobronchial tree and surrounding tissues compared with 3D-bTFE19.3D-bTFE also can demonstrate tracheobronchial anomalies but the image quality is less optimal. 3D-bTFE has higher sensitivity to B0 field inhomogeneity and signal loss artifacts can be present at air-tissue interface and at the periphery of the field-of-view; thus, the trachea and bronchus, especially upper portion of trachea are not clearly observedIn this study, predominant causes of tracheobronchial stenosis were vascular abnormalities which included double aortic arch, right aortic arch with mirror-image branching, right aortic arch with aberrant left subclavian artery or posterior PDA or ligament, left aortic arch with right descending aorta, innominate artery compression syndrome, left pulmonary artery sling, absent pulmonary artery valve, enlarged left atrium and dilated ascending aorta. MRI can also be used to evaluate the other associated cardiac defects and cardiac function through cine MRI and phase contrast sequences.There are some limitations in our study. First, there is a potential selection bias to this retrospective study and the expected frequency of these tracheobronchial anomalies in our group was higher. Second limitation is that there was no surgical/pathologic results to compare, but CT can be a good standard reference because of its advantage in demonstrating of lung and tracheobronchial tree.3D-TFE is a useful MRI sequence for the demonstration of the tracheobronchial tree and the diagnosis of tracheobronchial anomalies in congenital heart disease. MRI can supply helpful tracheobronchial information for preoperative strategies and postoperative follow-up.This retrospective research study was compliant with the Health Insurance Portability and Accountability Act and was approved by our tertiary care institutional review board. From December 1, 2013 to June, 2016, 75 patients with CHD (45 boys and 30 girls) who underwent both cardiac MRI and MSCT cardiac/chest examinations were enrolled. Their parents have signed informed consent before all the examination began. Patient ages ranged from 1.4 months to 134 months and the median age was 10.4 months. Cardiac MRI was performed to provide preoperative information about cardiovascular anatomy or/and cardiac function. MSCT was also performed either before or after the cardiac MRI examination. The time between the MSCT and cardiac MRI examination was 3.77 ± 7.08 months. MSCT examinations include ECG-gated cardiac CT (CCT), non-ECG-gated CT angiography (CTA) with intravenous contrast or CT of the chest (chest CT) without contrast.All MRI examinations were performed on a 1.5 T clinical MRI system  with a 16-channels torso or 8-channels cardiac phased-array coil. Two-dimensional cine balanced turbo field echo (2D cine b TFE), ECG-triggered 3D-bTFE, 3D-TFE with respiratory navigator, and contrast enhanced magnetic resonance angiography (CE-MRA) were performed. 2D cine bTFE, 3D-bTFE and CE-MRA were performed to evaluation cardiovascular anomalies and cardiac function. The ECG-triggered 3D-TFE sequence with respiratory navigator was performed on patients to evaluate the tracheobronchial tree. The acquisition parameters of these 4 sequences were summarized in Table All patients also underwent either low dose chest CT or CCT, or CTA before or after MRI examination. MSCT with 16 slices  or 64-slice high definition CT scanner .The parameters for CCT using the 64-slice CT scanner with prospective scanning mode was as follows: step-and-shoot axial scanning and a collimation of 64 × 0.625 mm with a scan field of view of 25 cm and gantry rotation time of 0.35 s; Tube voltage of 80–100 kV and tube current of 35–70 mA. All scans were reconstructed using 80% ASIR  algorithm.The parameters for CTA using 16 slice MDCT with non-ECG triggering scanning mode were as follows: 120 kV, 100–200 mA, 0.625 collimation 5.62 mm/s table speed, rotation speed 0.5 and 0.3-mm reconstruction.To decrease contrast medium-related artifacts and to achieve homogeneous contrast enhancement, MSCT was performed in the caudocranial direction when an arm vein was used and craniocaudal direction when a lower extremity vein was used. The injection rate using a power injector was 0.8–2.5 ml/s according to the scan range. The scan delay was 13–18 s depending on the site of injection or using bolus tracking. Non-ionic contrast agent (2 ml/kg), Iopamidol 370 mg/ml , was administered.The parameters for chest CT on 16-slice CT scanner were as follows: 120 kV, 50 mA, 7.5 mm, Collimation, pitch 1.375: 1, and 1.25 mm reconstruction.The 3D datasets of MRI and CT were processed on an offline EWS workstation , and Advantage Windows workstation 4.2–4.6  with maximum intensity projection (MIP) and minimum intensity projection (MinIP) algorithms and volume render techniques.20. The radiologists also independently evaluated image quality of 3D-TFE and 3D-bTFE based on reconstructed MinIP images on a five-point scoring system: 5 = excellent anatomical clarity and image quality ; 4 = good anatomical clarity and image quality (minor respiratory motion artifact); 3 = fair image quality ; 2 = poor image quality (inadequate delineation between the airway and the surrounding tissue), and 1 = non-diagnostic image quality. Scored over 3 were considered sufficient for diagnosis.The anatomy of the tracheobronchial tree and its anomalies in 75 patients were blindly evaluated by two pediatric radiologists with 14-years (Q. Wang) and 13-years experiences (A.M. Sun) in thoracic imaging, and the reviewing process was consensus reading by these two reviewers. We defined the local or long segment tracheobronchial stenosis and did not define the severity of stenosis . More than one-third of the tracheal length is called long-segment stenosis; otherwise it is local or short-segment stenosiskappa coefficient. A value of 0.8 or above indicates excellent agreement, 0.6–0.8 good agreement, 0.4–0.6, fair agreement and <0.4 poor agreement. Comparing the CT findings, the sensitivity, specificity, PPV, NPV, PLR and NLR of 3D-TFE and 3D-bTFE for the detection of tracheobronchial anomalies were evaluated for each observer using the consensus reading of the MSCT findings as the standard reference. Statistical analysis was performed using SPSS version 19.0 . A P value < 0.01 indicated statistical significance.Quantitative data were expressed as means ± standard deviations, and categorical data were given in proportions and percentages. The inter-modality agreement for tracheobronchial anomaly findings was tested by the The data that support the findings of this study are available from corresponding author upon reasonable request.This study was approved by the review board of Shanghai Children’s Medical Center Ethics Committee. ID nr: SCMCIRB-K2016009. This retrospective research study was compliant with the Health Insurance Portability and Accountability Act and all patients and/or guardians have signed informed consent before all the examination began.Supplementary information


Selection of residents is an extremely important process for a residency program as it determines the quality of patient care as well as the academic and research progress of the program. Most studies have focused on the relationship between the national board exam scores, such as the United States Medical Licensing Examination (USMLE) and the subsequent clinical performance of the selected residents. However, a low to moderate correlation has been found between board scores and residency performance.Additionally other studies have assessed the residency program directors’ opinions about the relative importance of various selection strategies. They found the top three common selection elements were clerkship grades, reference letters, and the USMLE Step 1 scores.There is no consensus as to which selection strategy is the most useful in choosing the candidates. Thus, the existing selection process is not ideal and has many shortcomings. For instance, it may not give an accurate representation of the interviewee as a medical resident.During the 2015-2016 interview season, the University of Toledo Internal Medicine Residency Program introduced a panel-style interview. Previously, the residency program relied on one-on-one interviews with various faculty members to evaluate candidates. Changing the interview style allowed an opportunity for multiple interviewers (the panel) to evaluate a residency candidate. Our panel members consisted of the program director/associate program director, a faculty member, and the chief resident. Also, under the one-on-one interview format, it allowed the interviewer greater access to beforehand knowledge of the candidate, which was changed under the panel interview style. Instead, the interviewers were blinded to the objective criteria, such as USMLE scores. After completion of the interview, the panel members individually scored the residency candidate using various subjective elements, such as communication skills, teaching potential, and professionalism. Subsequently, these scores were used by the residency program to calculate the final rankings of all the candidates during the interview season.The panel-style interview approach was designed to emphasize a balanced assessment tool that takes into account both the objective and subjective aspects of the residency candidate. We found that the quality of medical school was the strongest objective factor in the final ranking of the candidates. We question whether this was due to our arbitrary scoring system that might give too much advantage to US programs over international schools. Likewise, evaluators might show some bias toward residency applicants from our medical school. Another strong factor was the subjective component of the scoring system, which came ahead of the USMLE scores. We considered the fact that since panel members were blinded to objective factors, they were less likely to anchor too heavily on USMLE scores and dismiss the other qualities revealed during the interview when it was time to score the candidate. Another pertinent finding was that the program director/associate program director, faculty members, and the chief resident scored the candidates similarly when assessing the subjective elements. This suggests the panel members were consistent in how they viewed the candidate’s professionalism, teaching potential, and communication skills.The residency interview process varies across different academic programs around the US and in other countries. Some residency programs may give more weight to the subjectively assessed potentials of the candidates.Conversely, some residency programs may rely more on the objective parameters, such as USMLE scores, quality of medical school, academic performance, and scholarly activity. In either case, interviewing candidates is a complex process for residency programs and should include a multitude of assessment tools. We believe our panel style interview approach to evaluate internal medicine residency candidates is the first step in the development of both a standardized and balanced assessment tool at our institution.The authors declare that they have no conflict of interest.


Targeting leukemia-initiating cells (LICs) is the key to eradicating leukemia and preventing its relapse. Recent studies have indicated that metabolic regulation may play a critical role in the maintenance of stemness in LICs, although the detailed mechanisms are poorly understood. Herein, we provide intriguing evidence showing that a glucose-responsive transcription factor, carbohydrate responsive element binding protein (ChREBP), served as a tumor suppressor rather than an oncogene, as previously described, to inhibit the development of acute myeloid leukemia by promoting the differentiation of LICs. Using an MLL-AF9-induced murine leukemia model, we demonstrated that the deletion of ChREBP resulted in the blockage of the differentiation of LICs and significantly reduced survival in ChREBP-null leukemic mice. However, ChREBP was not required for the normal repopulation abilities of hematopoietic stem cells. ChREBP promoted leukemia cell differentiation through the direct inhibition of RUNX1 or the transactivation of TXNIP to downregulate the RUNX1 level and ROS generation. Moreover, knockdown of ChREBP in human leukemia THP1 cells led to markedly enhanced proliferation and decreased differentiation upon PMA treatment. Collectively, we unraveled an unexpected role of ChREBP in leukemogenesis, which may provide valuable clues for developing novel metabolic strategies for leukemia treatment. Acute myeloid leukemia (AML) is mainly caused by the accumulation of a variety of genetic mutations, such as chromosome translocations, deletions and point mutations. Mixed-lineage leukemia (MLL) rearrangements commonly occur in many subtypes of AML and infant acute lymphoblastic leukemia . Recent In the past decades, multiple oncogenic pathways have been found to be involved in metabolic alterations during cancer development. For example, PI3K/AKT/mTOR signaling is one of the most commonly altered pathways in human cancers, including leukemia and lymphoma. Studies have shown that the dysregulation of many genes that are involved in glycolysis or oxidative phosphorylation, such as glucose transport protein 1 (GLUT1), glucose-6-phosphate dehydrogenase (G6PD) and isocitrate dehydrogenase 1/2 (IDH1/2), leads to a high risk of the occurrence of leukemia or other tumors –11. AlthCarbohydrate response element binding protein (ChREBP) is a large transcription factor containing approximately 850 amino acids that is highly conserved among species . ChREBP Herein, we demonstrate that ChREBP is highly upregulated in mouse AML cells (as well as LICs) and acts as a tumor suppressor, rather than as an oncogene as previously reported, to inhibit the differentiation of LICs from a MLL-AF9-induced murine AML model. ChREBP deletion led to notably accelerated leukemia progression. ChREBP collaborated with TXNIP and RUNX1 to promote LIC differentiation. This study unravels a differential role of ChREBP in leukemia development compared to that in other solid tumors, which may open a new avenue for the treatment of leukemia or other types of cancers.+ leukemia cells or YFP+Mac-1+c-Kit+ LICs from a MLL-AF9-induced AML model, as determined by RT-PCR  recipient mice  and a notably increased cell number in colonies derived from ChREBP-null leukemia cells isolated from secondary recipients, indicating their enhanced clonogenic potential , as measured by quantitative RT-PCR in ChREBP-null LICs Figure . ConsistMoreover, several known targets important for lipogenesis, including FAS, ACC1, SCD1 and TXNIP (but not ACL), were markedly downregulated in ChREBP-null LICs. Many studies indicate that lipogenesis is required for the growth of cancer cells, which contradicts the marked decrease of FAS, ACC1 and SCD1 (genes that enhance lipogenesis) and accelerated leukemia development upon ChREBP deletion reported here. Interestingly, we found that TXNIP  was dow+ BM leukemia cells and LICs had relatively lower ROS levels compared to the WT controls  containing a conserved ChREBP-binding site Figure  and demoin vitro, even two days after knockdown , THP1 , HL60  and U937  were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% FBS (Hyclone). All the cell lines were from ATCC.− fetal liver cells were isolated from WT and ChREBP-null mice and infected with MLL-AF9 retroviruses by spinoculation in the presence of 4 μg/mL Polybrene. The cells were cultured overnight in DMEM with 10% FBS, 20 ng/mL SCF, 20 ng/mL IL-3 and 10 ng/mL IL-6, followed by another round of spin infection. The infected cells  were transplanted into lethally irradiated  C57BL/6 mice by retro-orbital injection. YFP+ bone marrow leukemia cells from primary transplanted mice were further isolated and injected into recipient mice for serial transplantation. For the rescue experiment, the retroviral plasmid MSCV-TXNIP-IRES-mCherry was used to transfect 293T cells, and the resulting retroviral supernatant was collected for spin infection with ChREBP-null leukemia cells, followed by retro-orbital injection into recipient mice.The MSCV-MLL-AF9-IRES-YFP-encoding plasmid  and the Peripheral blood was collected by retro-orbital bleeding, and bone marrow cells were isolated from the femurs and tibias of leukemic mice. Flow cytometry and cell cycle analyses were performed as we described previously . BrieflyEqual numbers of bone marrow leukemia cells or leukemia cell lines were collected for further immunoblotting. Whole cell lysates were electrophoresed on 8–10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% non-fat milk/TBS and incubated with primary antibodies at 4°C overnight. The following antibodies were used for blotting: anti-TXNIP (Proteintech), anti-ChREBP , anti-β-actin (Sigma), anti-HA (Sigma) and anti-StrepII (Genescript).THP1 cells infected with shRNA targeting ChREBP were treated with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) for 48 h as previously described [The lentiviral vector GIPZ was used to express shRNAs designed to target ChREBP . The PCR reactions were performed according to the manufacturer's protocol. The mRNA level was normalized to the level of the β-actin RNA transcripts. The primer sequences used are shown in Total bone marrow cells, HSCs, YFP5 WT or ChREBP-null AML cells per well were seeded in 96-well XF96 plates coated with BD Cell-Tak (BD Biosciences) in unbuffered DMEM and incubated at 37°C for pH stabilization. Analyses were performed both under basal conditions and after the injection of oligomycin (2 μM), glucose (10 mM) and 2-DG (100 mM).The ATP content was determined using an ATP Bioluminescence Assay Kit HS II (Roche) according to the manufacturer's protocols. Lactate generation was measured using the Seahorse XF96 extracellular flux analyzer as previously described, with minor modifications . Briefly® Multi Instrument). Alternatively, the luciferase activity of THP1 cells or 293T cells overexpressing ChREBP and plenti-TXNIP-GFP was measured. To evaluate the transcriptional activation of RUNX1 and GATA2 by ChREBP or TXNIP, the promoter regions of RUNX1 and GATA2 were incorporated into a pGL4 vector and analyzed with a luciferase reporter system as described above.The transcriptional activation of TXNIP by ChREBP was evaluated using a constructed lentiviral luciferase reporter vector (kindly provided by Guoqiang Chen at Shanghai Jiao Tong University School of Medicine), plenti-TXNIP-GFP, which contains the conserved ChREBP-binding site, as previously described . THP1 ce6 ChREBP-overexpressing (with a StrepII tag) THP1 cells were collected and cross-linked by adding formaldehyde at a final concentration of 1%. The samples were sonicated with a Q700 sonicator six times (5 sec on and 10 sec off for each round of sonication). ChREBP was immunoprecipitated with Strep-Tactin beads (IBA). DNA fragments were purified using a Qiagen PCR purification kit and quantified by semi-quantitative PCR with primers for the TXNIP promoter, listed in Chromatin immunoprecipitation (ChIP) assays were performed as described previously . A totalt test, and the statistical significance was set at p < 0.05. The survival rates of the two groups were analyzed using a log-rank test.The data are expressed as the mean ± SEM. The data were analyzed with Student's


The Strengthening and Stretching for Rheumatoid Arthritis of the Hand (SARAH) is a tailored, progressive exercise program for people having difficulties with wrist and hand function due to rheumatoid arthritis (RA). The program was evaluated in a large-scale clinical trial and was found to improve hand function, was safe to deliver, and was cost-effective. These findings led to the SARAH program being recommended in the UK National Institute for Health and Care Excellence guidelines for the management of adults with RA. To facilitate the uptake of this evidence-based program by clinicians, we proposed a Web-based training program for SARAH (iSARAH) to educate and train physiotherapists and occupational therapists on delivering the SARAH program in their practice. The overall iSARAH implementation project was guided by the 5 phases of the analysis, design, development, implementation, and evaluation (ADDIE) system design model.The objective of our study was to conduct the first 3 phases of the model in the development of the iSARAH project.Following publication of the trial, the SARAH program materials were made available to therapists to download from the trial website for use in clinical practice. A total of 35 therapists who downloaded these materials completed an online survey to provide feedback on practice trends in prescribing hand exercises for people with RA, perceived barriers and facilitators to using the SARAH program in clinical practice, and their preferences for the content and Web features of iSARAH. The development and design of iSARAH were further guided by a team of multidisciplinary health professionals (n=17) who took part in a half-day development meeting. We developed the preliminary version of iSARAH and tested it among therapists (n=10) to identify and rectify usability issues and to produce the final version.The major recommendations made by therapists and the multidisciplinary team were having a simple Web design and layout, clear exercise pictures and videos, and compatibility of iSARAH on various browsers and devices. We rectified all usability issues in the preliminary version to develop the final version of iSARAH, which included 4 short modules and additional sections on self-assessment, frequently asked questions, and a resource library.The use of the ADDIE design model and engagement of end users in the development and evaluation phases have rendered iSARAH a convenient, easy-to-use, and effective Web-based learning resource for therapists on how to deliver the SARAH program. There is also huge potential for adapting iSARAH across different cultures and languages, thus opening more opportunities for wider uptake and application of the SARAH program into practice. Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that presents with pain, inflammation, stiffness, and reduced muscle strength, joint movements, and joint function ,2.Joints of the hands and wrists are very commonly affected in people with RA ,3, resulDue to the success of the program and the NICE recommendations, we are now aiming to disseminate the evidence-based SARAH program to facilitate its use in clinical practice. In the original clinical trial, therapists attended a face-to-face training session  to learn how to deliver the SARAH program. Following the publication of the SARAH clinical trial results, all the patient and therapist materials required to deliver the SARAH program were made available for health care professionals worldwide downloadable from the Oxford Clinical Trials Research Unit (OCTRU) website .However, we recognized the need for a knowledge dissemination tool with the potential to facilitate wider and systematic uptake of the SARAH program by physiotherapists and occupational therapists and its implementation in clinical practice. We, therefore, proposed a free Web-based training program for SARAH, iSARAH , to servThe iSARAH implementation project is based on the analysis, design, development, implementation, and evaluation (ADDIE) model, one of the common instructional system design models used for constructing Web-based programs -24.The analysis stage comprises defining the problem, identifying the target knowledge users, and looking for possible solutions to bridge the knowledge-action gap and user-specific needs for the dissemination tool. In the context of the SARAH program, the knowledge-action gap is the evidence-based SARAH program (current knowledge) and its application in practice (action). The targeted users are the physiotherapists and occupational therapists who routinely treat and prescribe hand exercises to people with RA. We proposed to bridge the knowledge-action gap by educating and training the therapists on the SARAH program with a knowledge dissemination tool (iSARAH).The design stage consists of finding ways to organize and present the content, identifying modes of delivery, and developing an evaluation plan of the dissemination tool. This stage involves conceptualizing and adapting the SARAH program to fit the Web-based iSARAH.The development stage involves building iSARAH, evaluating its usability issues, and refining iSARAH to develop the final version.The implementation stage involves making iSARAH available for NHS therapists.The evaluation stage will include evaluation of learning outcomes such as knowledge, attitudes, intention to implement and user satisfaction with iSARAH, and evaluation of actual use of the SARAH program by iSARAH-trained therapists in real-world settings.Here we describe the first 3 phases of the iSARAH implementation project.Specific objectives of this phase were (1) to explore routine exercise prescription practices and outcomes use among therapists who treat people with RA affecting the hands and wrists, (2) to identify barriers and facilitators to implementing the SARAH program, and (3) to collect therapists’ opinions and preferences on the design, content, and features of iSARAH.A convenience sample of physiotherapists and occupational therapists of different countries who downloaded the SARAH program materials from the OCTRU website and gave permission to be contacted by the SARAH team were considered eligible for participation in the SARAH survey. Willingness to provide consent for taking part in the survey was the other inclusion criterion.We developed a survey questionnaire  that focThe survey protocol was reviewed and approved by the medical sciences Inter-Divisional Research Ethics Committee at the University of Oxford, Oxford, UK (reference number R43362/RE001). The SARAH survey was developed using LimeSurvey (LimeSurvey GmbH), an open source survey tool, and was hosted by OCTRU, University of Oxford.Specific objectives of this phase were (1) to design a paper prototype of iSARAH, and (2) to gain feedback from a multidisciplinary group of health professionals and to agree on the content, delivery methods, frequently asked questions (FAQs), and the navigation, layout, and visual appeal features of iSARAH.The SARAH research team and information technology experts mapped the SARAH program from the SARAH clinical trial to a 3- to 4-hour Web-based training package for therapists and designed a paper prototype. We proposed a half-day meeting with rheumatology clinicians, researchers, and technology experts based on their convenience and availability to attend the meeting. The purpose of this meeting was to gain collective feedback on the prototype and the survey findings to finalize the design of iSARAH. The paper prototype was presented at a half-day multidisciplinary team meeting (n=17) involving a rheumatologist (n=1), occupational therapists and physiotherapists , SARAH trial researchers (n=4), and information technology experts (n=2).Specific objectives of this phase were (1) to develop the iSARAH website, (2) to gain end user feedback on the usability, usefulness, ease of use, and confidence in using iSARAH, and (3) to rectify usability issues and further refine iSARAH prior to its implementation.This phase involved building iSARAH (preliminary version) and evaluating its usability, usefulness, and ease of use and user confidence ,26. The NHS hand therapists  who were treating people with RA and lived within 2 hours of travel to Oxford were considered eligible for participation in the usability testing. Willingness to provide signed consent was the inclusion criterion. We invited volunteers via the Centre for Rehabilitation Research in Oxford Twitter page and the online community forum of the Chartered Society of Physiotherapy.Based on the available evidence that 80% of usability issues can be identified by testing with 5 participants and that 95% can be identified with 9 participants ,28, we pWe coordinated individual appointments to attend usability sessions through telephone calls and conducted the sessions at the Botnar Research Centre, University of Oxford. Before evaluation, participants provided signed consent and completed a series of demographic questions. The usability testing procedure was then explained emphasizing that the session was about evaluating iSARAH and not the user. Each session took approximately 90 minutes. The usability testing involved the following procedures.The procedure was facilitated by 1 of the members of the SARAH implementation team. Participants were asked to log on to the iSARAH website by registering with test usernames and passwords. They were then asked to navigate through the website, starting from the home page. They were simultaneously encouraged to talk about what they felt, saw, or thought while browsing. The facilitator observed and took notes as participants were asked to verbalize their thoughts. When participants had difficulties in verbalizing, they were encouraged by a “keep talking” signboard and were minimally assisted with prompts (only when required) by the facilitator. All think-aloud sessions were audio recorded.We used the Computer System Usability Questionnaire (CSUQ)  to evaluWe measured iSARAH usefulness on a 5-point Likert scale .We measured overall ease of use on a 5-point Likert scale .We measured confidence in using iSARAH on a 5-point Likert scale .Using a semistructured interview guide, we asked participants about their experiences in navigating iSARAH. Interviews were conducted for approximately 10 to 15 minutes and were audio recorded. We summarized users’ comments on iSARAH by listening to the audio files and cross-checking a second time.We sent SARAH survey invitations to a total of 102 physiotherapists and occupational therapists who had downloaded the SARAH program materials. Pain, self-reported hand function, joint range of motion, stiffness, grip and pinch strength, and joint deformities were more commonly evaluated as part of their current practice. Performance-based hand function, 28-joint Disease Activity Score, and activities of daily living were the least evaluated outcomes.The most common type of exercise prescribed by therapists was active range of motion exercises. Strengthening exercises were also frequently used, as were tendon gliding exercises. Nerve gliding, passive, and isometric exercises were much less commonly prescribed.Self-management strategies, joint protection, and splinting were more commonly prescribed than thermotherapy, therapeutic gloves, work support, advice on activities of daily living, and electrotherapy.On average, therapists had 4 sessions with their patients . The frequency of review sessions was mostly either once every 15 days, reported by 11 therapists, or every 1 to 2 months, reported by 9 therapists.Most therapists used exercise sheets and review appointments to encourage adherence with home exercise programs. Exercise diaries, exercise contracts, and telephone reminders were less commonly used.About 74% (n=26) of the therapists delivered the SARAH program in their clinical practice, and on average had prescribed the program to 17 (SD 22) of their patients since downloading the materials. More than 50% (17/26) of the therapists who delivered the SARAH program did not find any aspect of SARAH that made it difficult to put into practice. They reported that the SARAH therapist manual, the exercise sheets with photographs, and the strong evidence base facilitated their use of the SARAH program in their daily practice. Other therapists reported issues with time, funding for exercise equipment, and inability to complete review assessments and exercise contracts.Therapists who did not use the SARAH program (n=9) reported a lack of appropriate patients to be prescribed the SARAH program, budget, time, and their routine prescription of hand exercises like the SARAH program as main reasons for nonimplementation. We asked therapists what they would like to see in a Web-based training program if one were available. Following the multidisciplinary team meeting, we identified the specific need to educate and train therapists on the behavioral support strategies and proposed a separate module on this topic. We agreed that a section addressing common questions that might be raised by therapists about the SARAH program in real-world settings should be included in an FAQ section of iSARAH. Attendees provided suggestions for framing these questions. Based on discussions about the iSARAH prototype and SARAH survey findings, the team suggested the following recommendations: (1) to provide weblinks within the text for additional information on a topic, for example, Splints in RA, (2) to provide a progression status bar to enable users to know where they are in the training, (3) to use consistent names for exercises, (4) to have a separate educational video on joint protection advice, (5) to have a separate module on behavioral support strategies, (6) to have SARAH exercises demonstrated through videos and photographs, (7) to have brief modules, (8) to have a plain layout and use optimal font sizes (14 point), (9) to have an official email support to address technical enquiries, and (10) to ensure iSARAH adapts across different types of Internet browsers and computers at NHS settings and other telecommunication devices.Specific recommendations were also made regarding the behavioral strategies module: (1) to provide examples of general goals relating to upper limb function to aid therapists with goal setting, and (2) to include model scenarios on filling in the personal exercise guide and Barriers and Facilitators form.A simple Web layout and designExercise photographs and videos with clear instructionsBrief training modulesA self-assessment sectionSimplified Strengthening and Stretching for Rheumatoid Arthritis of the Hand (SARAH) patient materialsInstructions on how to complete the personal exercise guide, exercise diary, and Barriers and Facilitators formDownloadable SARAH program materialsOnline support for technical queriesLinks for patients to access information or complete outcomes onlinePowerPoint teaching materialsCompatibility with different browsers, such as Internet Explorer and Google Chrome, and responsiveness in different devices, such as mobiles and tabletsMake it clear in iSARAH that the SARAH program is flexible and will be feasible to complete at the user’s convenience.Send monthly email reminders to iSARAH-trained therapists.Signpost therapists and their patients to resources needed to deliver the program, which could, ideally, be purchased at a discounted rate .Provide multiple hard copies of the SARAH patient materials at no cost to iSARAH-trained therapists for use in clinical practice, if required.Demonstrate high credibility by incorporating information about the SARAH research team and all SARAH peer-reviewed publications.Propose pain and self-reported hand function as the main outcomes for the evaluation phase.To facilitate effective implementation of the SARAH program by iSARAH-trained therapists in actual practice, we also discussed ways to minimize major implementation barriers reported in the survey . Clinicians who had worked on the SARAH clinical trial raised some issues with the original forms used in the trial, and they proposed ways to streamline these forms to make them easier to use.iSARAH was built on a Moodle platform  by the OCTRU information technology team, customized and styled using the Essential Theme add-on. An overview of iSARAH (preliminary version) is provided below.The landing page introduced the iSARAH with a brief statement about the purpose of the website, site contact information, privacy policy, and the modules. Other features included a 2.5-minute preliminary iSARAH promotional video and a prominent widget for logging on to the training.Module 1 covered clinical aspects of RA, benefits of exercises in RA, UK guidelines in the management of RA, and information about the SARAH clinical trial.Module 2 covered development and physiological principles of the SARAH program, behavioral support strategies, and instructions on how to deliver the SARAH program.Module 3 covered the self-assessment.Module 4 included FAQs to inform the delivery of the SARAH program in different practice settings and patient scenarios.All text materials required to deliver the SARAH program  and additional reference documents, such as SARAH trial publications, were archived in the resource library.A combination of text, photographs, tables, and videos was used to deliver the training. Preliminary videos were produced for iSARAH promotion and instruction purposes of the training.A simple Web layout was used consistently across modules to reduce distraction and information overload.One of the major usability issues we observed was the difficulty in navigating from the end of one module to the next , as there were no direct buttons to take users to the following module. Instead, participants had to click the respective module tabs on the top of the screen to navigate between modules or to proceed to the next module. We also noticed that some additional tabs appearing within the Moodle platform were confusing for the participants.Hyperlinks to reference documents such as SARAH trial publications and patient materials were reported to be repetitive and distracting. Participants said that photographs showing RA hands and activities of daily living, and other illustrations, did not add to iSARAH but instead occupied screen space and led them to frequently scrolling down to read the whole page. In the self-assessment module, when participants entered an incorrect response to a question, they couldn’t find a feature to signpost to the correct response in the respective module. They also reported that information about the SARAH team on the home page was not adequate.The CSUQ showed that participants overall found iSARAH simple, easy to use, and easy to understand, and they were satisfied in using it . There wIn general, users found that iSARAH was a detailed and helpful learning resource for therapists. The most common comments were that participants liked the Web layout, tabs for modules, exercise videos, and the whole content. Some key suggestions provided were to create videos of good sound quality, and to remove excess text and photographs to keep the information relevant and clear.We revised iSARAH to address all major usability issues identified from the think-aloud procedure and interviews . We prodPrior to official launch on April 3, 2017, we tested and activated the following features: (1) online user registration page, (2) online feedback questionnaire on perceived usefulness, satisfaction, and intention to use the SARAH program in future practice, and (3) download option for the training completion certificate.The overall purpose of this paper was to present how we developed a Web-based implementation tool (iSARAH) and produced the final version suitable for implementation. The strength of this work is that it followed a recognized model for the construction of Web-based programs -24.Engagement with users through the SARAH survey allowed us to identify current practice and learning needs to ensure iSARAH was fit for purpose. From the survey, we established that the exercises included in the SARAH program were commonly used by therapists ,7,30,31 Engagement with users continued during the design phase with a face-to-face meeting, as well as carrying out usability testing. Usability testing was essential to producing a user-friendly website that could be deployed for implementation. We believe this has resulted in a flexible learning experience for users, which is easy to navigate with unlimited access. We included FAQs and self-assessment to ensure that therapists have adequate training and skills to efficiently apply for the SARAH program in actual practice.The next step is to evaluate the impact of iSARAH training on actual implementation of the SARAH program, including the impact on knowledge and skills of therapists, implementation rates, and patient outcomes. We know from our previous work  that traThis study has some limitations. First, we neither used observational analysis with video recordings in the think-aloud procedure to observe users’ interactions with iSARAH, nor conducted a systematic qualitative analysis of participants’ interviews. Second, the CSUQ and Likert scales have not been tested for reliability and validity in the target population. Hence, the range of scores should be interpreted with caution. Third, the SARAH survey participants were familiar with the SARAH program and hence their responses were prone to the risk of volunteer bias. Additionally, with a low consent rate , the survey findings are at the risk of nonresponse bias from people who did not participate or respond. Fourth, we did not employ iterative cycles of usability testing—that is, consecutive cycles of testing until the point when no further usability issues were identified—but we used the feedback from all participants in a one-off cycle to refine iSARAH.Evidence-based therapies have been found to be poorly disseminated into routine practice . Some ofIn our next steps toward opening more opportunities for wider uptake and application of the SARAH program into clinical practice, there is also a huge potential for adapting iSARAH across different cultures and languages across the world.To our knowledge, iSARAH is the first Web-based learning resource for therapists on an evidence-based hand exercise program. A systematic design approach by using the ADDIE model and involving end users has been successful in developing a user-centered iSARAH.Our ongoing work on the impact evaluation among therapists who completed iSARAH and a service evaluation in people treated by SARAH-trained therapists will provide more insights on the uptake of the SARAH program in actual practice.


An earthquake struck Yushu in Qinghai province of China on April 14, 2010, causing 2698 deaths and 12,135 injuries.The present study aimed to assess the health status, and associated determinants, of child survivors in the epicenter of the Yushu earthquake 6 years after the event.A cross-sectional survey was performed among students from two junior schools in Yushu County. Descriptive statistics, t-tests, ANOVA, Wilcoxon rank sum tests, Kruskal-Wallis H tests and stepwise linear regression analysis were used for data analysis.The mean scores onmental component summary (MCS)and physical component summary (PCS) were 42.13 (SD 7.32) and 42.04 (SD 8.07), respectively. Lower PCS in the aftermath of an earthquake was associated with being trapped/in danger, injured to self, receiving no escape training while lowerMCS in the aftermath of an earthquake was associated with a lower grade level, not living with parents, fear during the earthquake, death in the family, and not receiving psychological counseling after the earthquake.In conclusion, the results of the present study help to expand our knowledge regarding the health status of child survivors 6 years after the Yushu earthquake. Our study provides evidence-based suggestions for specific long-term health interventions in such vulnerable populations. Not only can natural disasters cause a devastating loss of life, homes, and livelihoods in an instant, they can also impose lingering threats to the physical and mental health of the survivors who must cope with post-disaster adversities for many years , 2. MoreThere is an emerging literature on the health status of earthquake survivors –7, with Child survivors are a vulnerable population whose health status is sensitive to external adverse events such as earthquakes. Several studies focusing on child survivors have shown that the HRQoLdecreases over time. Enabling children who have survived disasters to achieve and maintain the same standards of health as other children and adolescents is a goal of the international community .In particular, for relatively remote plateau areas like Yushu, the health status of child and adolescent survivors should be continuously considered. Therefore, the present study aimed to assess the health status, and associated determinants, of child survivors in the epicenter of the Yushu earthquake 6 years after the event, to provide evidence-based suggestions for specific long-term health interventions in such vulnerable populations.A cross-sectional survey was conducted in August 2016, at which time 6 years had passed since the event . The capThe survey was conducted by 12 undergraduates from the Second Military Medical University. The Project Leader conducted a centralized training in Shanghai for the 12 investigators on August 12, 2016, briefed the prospective respondents about the details of the study, and answered questions related to the study. The 12 researchers were divided into 6 groups and were dispatched to Yushu County to conduct the investigation from the 13th to the 26th of August, 2016. The survey took place in the school classrooms. Given the limited comprehensive ability of the middle school students, each groupof researchers briefed the prospective respondents, providing details of the study, and answered related questions. Approval was obtained from the school principal who made an announcement regarding the study to the students. The study also obtained approval from the ethics committee of Second Military Medical University, and all participants voluntarily agreed to participate in our survey.The collected social and demographic information included sex, age, ethnicity, religious faith, chronic disease history, residential area, family resident population, main source of income for the family, whether living with parents or not, whether an only child or not, and family type. Students “not living with parents” refer to the left-behind children .Earthquake-related experiences were assessed using an inventory adapted from a commonly used earthquake exposure scale. The items included ‘being trapped or in danger’, ‘house damaged’, ‘family property damaged’, ‘injured’, ‘family member injured’, ‘lost family member or friend’, degree of fear during the earthquake, degree of fear, etc. .The health status of the students was assessed using the acute version of the Chinese version of the Short Form-12 (SF-12), which is a practical, reliable, and valid scale adapted from the 36-item Short Form health survey (SF-36). The Chinese version of the SF-12 has been validated for Chinese adolescents . The parp values <0.05, multiple comparison tests were conducted using the least significant difference(LSD) test. A multiple linear regression including all the variables was used to find the factors independently associated with PCS and MCS scores [p < 0.05 was considered statistically significant.All analyses were performed using the Statistical Package for the Social Sciences (SPSS) Version 11.0 . Descriptive statistics  were first calculated. Subsequently, t-tests (for two-group comparisons) and analyses of variance  were used to evaluate differences in continuous variables when the data were normally distributed and in accordance with assumptions regarding the homogeneity of variance; otherwise, non-parametric methods were used .For ANOVAs with a S scores . MeanwhiS scores . A p < 0n = 252, 42.64%) reported that their houses had been damaged during the earthquake. Similarly, less than half of the students  reported that their family experienced property loss due to the earthquake. A total of 318 participants (53.81%) had received psychological escape training and counseling.Social-demographic characteristics and earthquake-related experiences are presented in Table P < 0.0001).In bivariate analyses (Table p < 0.001), ‘house damaged’ (p = 0.0314), ‘injured’ (p = 0.0002), ‘family member injured’ (p = 0.0442), and not receivingescape training (p = 0.0002). Multiple stepwise regression analyses (Table p = 0.0001), ‘injured’ (p = 0.0323), and not receiving escape training before the earthquake (p < 0.0001).The mean PCS score was 42.13 (SD 7.32),which was significantly lower than the PCS of the general adolescent population in China .In bivariate analyses (Table p < 0.0001) and were positively associated with a higher grade level (p < 0.0001), living with parents (p = 0.0021), and receiving psychological counseling (p = 0.0320). Multiple stepwise regression analyses (Table p = 0.0006), not living with parents (p = 0.0084), fear during the earthquake (p = 0.0081), death in the family (p = 0.0005), and not receiving psychological counseling after the earthquake (p = 0.0275).The mean MCS score was42.04 (SD 8.07), which was significantly lower than the MCS of the general adolescent population in China  . Since tThe current study has a few limitations to discuss. First, due to the study’s exploratory cross-sectional nature, it is difficult to conclude to what extent the earthquake had an effect on those who experienced it, as no pre-disaster or short-term after-disaster comparison data was available. Second, although the study was conducted adequately by trained investigators, data collection relied solely on the retrospective self-reports of the survivors 6 years after the Yushu earthquake, which can be subject to recall and desirability biases. Despite these limitations, the present study is one of only a few cross-sectional studies focusing on the long-term health status and associated risk factors in child survivors following an earthquake in rural China.In conclusion, the results of the present study help to expand our knowledge regarding the health status of child survivors 6 years after the Yushu earthquake. The results suggest the importance of greater vigilance and long-term attention to the children who lived in the rural plateau area and were most affected by the earthquake. This study provides important information regarding monitoring the long-term course of physical and mental health recovery of adolescents after earthquakes, and is of great significance for public awareness and the sensitivity towards those who were the most affected. Specific care, earthquake escape training, and long-term psychological interventions should be provided to adolescent survivors with relatively low health status, including those who were injured in the earthquake and those who were not living with parents.


Advanced subspecialty training in reproductive endocrinology and infertility (REI) entails a competitive application process with many data points considered. It is not known what components weigh more heavily for applicants. Thus, we sought to study the REI fellow applicant and compare 1) those who apply but do not receive an interview, 2) those who receive an interview but do not match, and 3) those who successfully match.curriculum vitae. Logistic regression models were constructed to determine variables that were predictive of being offered an interview in our program and of matching in any program.This retrospective cohort study was conducted at a single REI fellowship program from 2013 to 2017. Academic variables assessed included standardized test scores and total number of publications listed on their There were 270 applicants, of which 102 were offered interviews. Interviewed applicants had significantly higher mean USMLE 1 and CREOG scores, as well as total publications and total abstracts. There was no difference in Step 2 and Step 3 scores or in number of book chapters. Of those interviewed, USMLE scores remained predictive of matching in any program; however, publications and scientific abstracts were no longer predictive.The decision to offer applicants interviews appears to be influenced by objective standardized test scores, as well as a threshold of academic productivity. These items are less predictive of matching once the interview process begins, indicating that other factors, such as performance during the interview day, may be more heavily weighted. There has been a focus on attracting and retaining high quality resident physicians in obstetrics and gynecology many of whom comprise and outstanding pool of applicants for post-resident fellowship training , 2 PositUntil the 2017 academic cycle, two professional organizations governed the REI fellowship match process. The Society for Reproductive Endocrinology and Infertility (SREI) served as a clearinghouse for applications by receiving the initial standardized applications and coordinating their distribution to programs participating in the match. Subsequently, the National Resident Matching Program (NRMP) served as a centralized place where applicants and programs ranked one another. Thus, those who initially applied through SREI did not always participate in the NRMP match process either because they ultimately elected not to pursue fellowship or, probably more commonly, because they were not invited to interview.According to the NRMP, the match rate for those who ranked at least one program in the match between 2012 and 2016 was 62.3–68.3% . This maTo that end, we sought to characterize the REI fellow applicant and compare 1) those who applied but did not receive an interview in our program, 2) those who receive an interview but do not match in any program, and 3) those who successfully match in any program.All applicant files for the Rutgers-Robert Wood Johnson/Thomas Jefferson fellowship program in REI were reviewed over a five-year period, including applications for fellowship training beginning July 2013 to July 2017. The fellowship transitioned from Rutger-Robert Wood Johnson to Thomas Jefferson in 2017 without change of core facility, faculty, and research core. This retrospective anonymized data review was IRB exempt. Applications were placed into three groups: those who applied but did not receive an interview in our program; those who received an interview in our program but did not match into any fellowship program; and those who ultimately matched into any program. At this fellowship institution, interviews are granted based upon program director and current fellow applicant review.In reviewing the applicant files, the following data were extracted if provided: medical school training; residency program training; Alpha Omega Alpha (AOA) Honor Society status; United States Medical Licensing Exam (USMLE) scores for Step 1, Step 2 Clinical Knowledge (CK), and Step 3; and scores on the Council on Resident Education in Obstetrics and Gynecology (CREOG) exams for the first, second, and third years of residency.Academic productivity was likewise assessed, according to the total number of manuscripts (including those in press and progress), the number of published citations in a peer-reviewed journal , the number of book chapter contributions, and finally, the number of scientific abstracts listed for presentation at scientific meetings.Given that many applicants during the study period exhibited some level of academic productivity, a subanalysis was performed to assess whether a “threshold of productivity” existed. Accordingly, the proportion of applicants having any publications, ≥1 publications and ≥2 publications were compared to those below this threshold.t-test, Mann-Whitney U test, Fisher’s Exact test, and Pearson’s Chi-square test where appropriate. Correlation tests for normally and non-normally distributed values were assessed using Pearson Correlation and Spearman’s Rho tests. A predication model was created based upon the variables which predicted the chance of matching. A P-value of <0.05 was considered significant.All statistical analyses were performed using SPSS statistical software . Each variable was assessed for normality utilizing histograms with overlying normality curve and Q-Q plots. Comparison of variables was accomplished with Student’s There were 270 applicants during the five-year application period (mean of 54 applicants per year). Of the 270 applicants, there were 211 US medical graduates and 59 foreign medical graduates (FMG); 47.1% of US medical graduates were offered interviews, as compared to only 5.1% of FMG. All FMG applicants offered an interview had trained in US obstetrics and gynecology residency programs.When looking at total applicants and residency program affiliation, applicants were categorized as belonging to an academic resident program if housed within a university, academic-affiliated program if the training was done at a hospital affiliated with a university, and community program if the training was done at a hospital unaffiliated with a university. In all, 70.4% came from academic residency programs, 9.6% from academic affiliated programs, and 20.0% from community programs.As for the geographic distribution of applicants, the residency program locations were noted to be in the Northeast, Midwest, South and West as defined by the U.S. Census. The majority of the applicants came from the Northeast which represented 49.3% of applicants. There were 17.4% of applicants from residency programs in the Midwest, 20.4% from the South, and 7.8% from the West Coast. There were 5.1% of those who applied the reported training outside of the United States.In total there were 102 applicants offered an interview for five fellowship position, one per year over the course of the analysis. The characteristics of those receiving an interview versus those who did not are listed in Table p = 0.001) and 3.1 [95%CI 1.1–8.3] (p = 0.028), respectively.The unadjusted models predicting the chance of being offered an interview are shown in Table Of the 102 offered interviews, 87 85.3%) matched into programs while 15 14.7%) did not. Demographics of those matching into a fellowship program as compared to those not matching amongst interviewed applicants are listed in Table % matched.7% did nOf note, when analyzing the academic productivity in terms of total number of manuscripts, published peer-reviewed manuscripts, book chapters, and scientific abstracts, these were not found to be predictive of matching once granted an interview. When analyzing the “threshold of productivity” for those who interviewed, no defined threshold for scientific abstracts threshold was predictive of matching.Matching into an REI training program is competitive, and mentoring applicants toward success can be challenging. According to data from the NRMP, the number of programs participating in the match has ranged from 36 to 40 over the period of 2012 to 2016 . Those pThis descriptive cohort shows that the typical REI applicant is scientifically productive and scores well on standardized tests. This generalization may not come as a surprise, but the specifics provided by these data can serve as a more concrete directive to potential applicants. For those hoping to be granted an interview, careful preparation in the years prior to application, with dedication to academic performance and standardized testing, is important. At our institution the decision to offer an interview is often based upon objective data such as USMLE scores and CREOG scores. Of note, there also appears to be an emphasis placed on a threshold of academic productivity.Interestingly, aside from USMLE scores, the objective data gleaned from applications seem to be somewhat less predictive of matching once the interview process begins. This suggests that, once an applicant is granted an interview, that other factors, such as letters of recommendation and interview day performance, may be more heavily weighted in the final ranking process.There are few published experiences in this realm. Uhlenhake et al. published on the experience of dermatopathology applicants . They noThere are several limitations to these data. They are gleaned from a single program based in the Northeastern region of the US. There may be regional differences in applicants who decide to apply due to geographic preferences. Further, this particular program was established in 2010 and thus is a newer training program. We also only had access to data from applicants who applied to our program, so these results may not be generalizable to all fellowships. Further, during the course of this data collection, the first round of application data was collected and distributed to programs through a centralized application process coordinated by the Society for Reproductive Endocrinology and Infertility (SREI). SREI does not publish the application data like the NRMP. Thus, we are not able to know how many applicants applied nationally and what percentage applied to this program.Finally, the overall match rate among those who interviewed at our program (85.3% of those who interviewed) was higher than the range from the NRMP (62.3–68.3%) over a similar timeframe. It is possible that these data characterize those fellow applicants more likely to match than the average fellow applicant and should be considered when mentoring future applicants.As a point for future research, while several of the objective factors are noted to be associated with matching into fellowship, it is not known how these factors predict academic productivity and clinical performance in fellowship and beyond. This would likely require a multi-center analysis of factors and performance and would an interesting area for research extension.This observational study provides a five-year single center summary of REI applicants. The field is competitive with only two-thirds of those who receive interviews at programs ultimately matching. The number who apply initially and ultimately match is far lower. This presents a challenge when it comes to mentoring those who desire to enter the field. These data help future applicants compare their academic performance to peers and characterize those who successfully navigate the process versus those who do not.


Endobronchial ultrasound(EBUS)-guided transbronchial biopsy(TBB) is the preferred diagnostic tool for peripheral pulmonary lesions(PPLs) and mastering this procedure is an important task in the training of chest physicians. Little has been published about the learning experience of physicians with this technique, particularly at an institutional level. We aimed to establish a learning curve for EBUS-guided TBB for PPLs at a medical center.Between 2008 and 2015, consecutive patients with PPLs referred for EBUS-guided TBB at National Taiwan University Hospital were enrolled. To build the learning curve, the diagnostic yield of TBB (plus brushings and washings) was calculated and compared. Meanwhile, lesion characteristics, and procedure-related features and complications were obtained to analyze associations with TBB yield and safety profile.A total of 2144 patients were included and EBUS-guided TBB was diagnostic for 1547(72%). The TBB yield was 64% in 2008 and reached a plateau of 72% after 2010. It took approximately 400 EBUS-guided procedures to achieve stable proficiency. Further analysis showed that improvement in diagnostic yield over time was mainly observed in PPLs, in cases in which the diameter was ≤2 cm or the EBUS probe could not be positioned within. Complication rates were low, with 1.8% and 0.5% for pneumothorax and hemorrhage, respectively.Even though EBUS-guided TBB is an easy-to-learn technique, it takes 3 years or around 400 procedures for a medical center to achieve a better and stable performance. In particular, the diagnostic yield for lesions without the probe within or those sized ≤2 cm could improve with time. Diagnosis of peripheral pulmonary lesions (PPLs) can be achieved with a variety of modalities, including bronchoscopy, video-assisted thoracoscopic surgery (VATS) and computed tomography (CT)-guided biopsy. Recent modifications of bronchoscopy with ancillary techniques, such as endobronchial ultrasound (EBUS), electromagnetic navigation and thin bronchoscopes,–6 have sA learning curve is a concept used to measure how soon one can be proficient in a skill. Understanding a single institutional or individual learning curve is of importance for setting standards, designing training programs and establishing competitive strategies. However, to the best of our knowledge, there is a paucity of such information about EBUS-guided TBB. Based on expert opinions, the guidelines recommend that each learner performs a minimum number of 40–50 EBUS-guided procedures for each learner to perform in order to achieve basic competence., 13 In rAccordingly, in this study, we aimed to establish the learning curve for EBUS-guided TBB for PPLs at a medical centre. At the same time, we sought to investigate whether the changes in EBUS performance over time were similar with patients with different clinical features.This retrospective study was conducted in accordance with the amended Declaration of Helsinki at National Taiwan University Hospital, a medical centre in northern Taiwan. We implemented the use of EBUS-guided TBB for PPLs in 2007, and beginning in late 2007, patients with PPLs no longer underwent conventional TBB. Following approval from the Research Ethics Committee of National Taiwan University Hospital, consecutive patients with PPLs referred for EBUS-guided TBB between January 2008 and December 2015 were enrolled in this study. In our institution, the minimum diameter of the PPLs considered for EBUS-guided TBB was 8 mm. A PPL was defined as a lesion that was surrounded by lung parenchyma and was not seen within the bronchial trees during conventional bronchoscopy. Written informed consent was obtained from all patients before the bronchoscopic procedure.The procedure was performed by chest fellows under the supervision of experienced pulmonologists. Conventional bronchoscopy with a 2.0-mm working channel  was first conducted to inspect the bronchial trees, after local anaesthesia with 5 ml of 2% lidocaine sprayed or nebulized into the upper airway mucosa and intramuscular injection of meperidine 50 mg if not contraindicated., 15 AddiThe primary outcome of interest was the diagnostic yield of EBUS-guided TBB of PPLs across the study period. The TBB was also considered diagnostic if the diagnosis of a PPL was achieved via bronchial washing or brushing during the same bronchoscopy session. As for other non-diagnostic PPLs, the final diagnosis was established by pathologic evidence from CT-guided or VATS biopsy, or other invasive diagnostic procedures, microbiologic analysis, serology testing or clinical follow-up. Other outcomes of interest included the time-varying changes in the diagnostic yield of EBUS-guided TBB of lesions with distinctive features. These features were pre-defined as those patient characteristics associated with a diagnostic EBUS-guided TBB of PPLs.Medical records were reviewed in detail for demographic data, lesion size, location and appearance on CT, ultrasonic probe position on EBUS images, final diagnosis, and bronchoscopy-associated complications. Lesion size was measured as the greatest axial diameter on CT scans. Lesion location was categorized into 5 anatomic lobes. If the PPL resided in 2 lobes of the lung, it was assigned to the lobe containing more than half of the lesion area. The lesion appearance was characterized as a solid, partly solid, pure ground-glass or cavitary opacity. The position of the EBUS probe was classified as within or adjacent to the PPL, as reported previously.2 or Fisher's exact test was used. With respect to the primary outcome, a multivariate logistic regression model was built to identify independent factors associated with a diagnostic EBUS-guided TBB. Variables with a p value of <0.2 were entered into the multivariate analysis. To investigate yearly trends in the diagnostic yield of TBB, we performed a linear-by-linear association χ2 test for categorical data. All analyses were conducted using SPSS for Windows, version 15.0  and a 2-tailed p value of <0.05 was regarded as statistically significant.Data were reported as No. (%) or mean±standard deviation, as appropriate. For comparisons between categorical variables, the χOver an 8-year period, a total of 2144 patients had been referred for EBUS-guided TBB of the PPLs, and 153 (7.1%) of them could not be localized by EBUS and EBUS-guided TBB was not attempted. The clinical characteristics of the study population are summarized in EBUS-guided TBB was diagnostic for 1547 (72%) of the PPLs. The diagnoses made by TBB are listed in The number of EBUS-guided TBB performed at our institution rose evidently between 2008 and 2015, and we observed a significant trend (p = 0.041) toward an increase in the diagnostic yield of TBB during the study period . The TBBFurther in-depth analysis showed that the diagnostic performance of EBUS-guided TBB was similar with PPLs sized >2 cm, with the ultrasonic probe within, and with both benign and malignant histologies across the study years . A signiTo our knowledge, this is the first study reporting on an institutional learning curve of EBUS-guided TBB in a large population and over a long period. The main findings of our study are as follows: (1) The diagnostic yield of EBUS-guided TBB for PPLs gradually improved over the first 3 years (or during approximately the first 400 procedures) and approached a plateau thereafter. (2) The improvement in TBB performance was mainly attributed to the increased TBB yield for PPLs with the probe adjacent to the lesions themselves and with an axial diameter of ≤2 cm. (3) The safety profile of EBUS-guided TBB for PPLs was excellent, as indicated by the extremely low risks of procedure-related complications. (4) The lesion size and histology, and ultrasonic probe position were significantly associated with the diagnostic yield of TBB.Although EBUS-guided TBB is a relatively easy-to-learn task, as reflected by a nearly 70% diagnostic yield for PPLs in the first year of our practice and excellent initial results from other study groups,, 2 its pAn interesting and important finding in this study is that an advance in the diagnostic yield of TBB was observed only in patients in which the EBUS probe was adjacent to the PPLs and with lesions sized ≤2 cm. PPLs with these features were found to be associated with a lower diagnostic yield of EBUS-guided TBB in the present and prior studies;, 16–18 tIn line with prior studies,, 16, 18 et al.[The overall diagnostic yield of EBUS-guided TBB in our institution falls within previously reported figures., 23–25 Set al. recentlySafety is an important issue for any kind of invasive procedures. For EBUS-guided TBB, pneumothorax and haemorrhage were the 2 most frequently encountered complications, in both our and other studies., 17, 24 This study has several limitations. First, a learning curve for EBUS-guided TBB in a medical centre setting may not be entirely representative of a more general patient population in other hospital settings. However, we wanted to share our experience to increase understanding of such information and to encourage others to share theirs. Second, the EBUS-guided TBB procedure was accomplished by a cooperative team consisting of chest fellows with different levels of experience in our institution, and they were supervised by experienced pulmonologists. Thus, the impact of operator expertise on the institutional learning curve could not be accurately assessed and we did not describe individual learning curves in this study. What we have described can be considered a real-world scenario in a teaching hospital. Third, other ancillary bronchoscopic techniques, such as using a guide sheath, an ultrathin bronchoscope, fluoroscopy, virtual bronchoscopic navigation, electromagnetic navigation, and rapid on-site evaluation,, 20 wereIn conclusion, the EBUS-guided TBB may be an easy-to-learn technique, but it takes 3 years or around 400 procedures for a medical centre to achieve a better and more stable performance. In particular, the diagnostic yield of PPLs with a size of ≤2 cm and without the probe within could be clearly improved with time. Meanwhile, in this study, the largest patient population to date, we have demonstrated and confirmed that probe position, and lesion size and histology are significant determinants for a diagnostic TBB of PPLs. Finally, the most appealing feature of EBUS-guided TBB is its excellent safety profile, which makes it the preferred modality for sampling a PPL.S1 File(XLSX)Click here for additional data file.