Dynamic contrast-enhanced MRI provides information on the heterogeneity of the synovium, the primary target of disease in children with juvenile idiopathic arthritis (JIA).To evaluate the feasibility of dynamic contrast-enhanced MRI in the wrist of children with JIA using conventional descriptive measures and time-intensity-curve shape analysis. To explore the association between enhancement characteristics and clinical disease status.n = 27) or inactive (n = 5). Outcome measures included descriptive parameters and the classification into time-intensity-curve shapes, which represent the patterns of signal intensity change over time. Differences in dynamic contrast-enhanced MRI outcome measures between clinically active and clinically inactive disease were analyzed and correlation with the Juvenile Arthritis Disease Activity Score was determined.Thirty-two children with JIA and wrist involvement underwent dynamic contrast-enhanced MRI with movement-registration and were classified using validated criteria as clinically active (P = 0.019), whereas time-intensity-curve shape analysis showed no differences. Juvenile Arthritis Disease Activity Score correlated moderately with enhancing volume (P = 0.484).Comprehensive evaluation of disease status was technically feasible and the quality of the dynamic dataset was improved by movement registration. The conventional descriptive measure maximum enhancement differed significantly between clinically active and inactive disease  contains supplementary material, which is available to authorized users. Juvenile idiopathic arthritis (JIA) is a chronic joint disorder in children with the synovium as the main target tissue for inflammation. The synovitis can eventually lead to structural damage in children with JIA, which obviously goes hand in hand with increased morbidity and decreased quality of life. JIA has a relapsing-and-remitting disease course, making adequate assessment of disease activity a real challenge and necessity, in order to provide tailored treatment for every patient. Currently, the distinction between active and inactive disease is based on a clinical assessment, but imaging techniques are becoming more and more valuable in this respect.Conventional contrast-enhanced MRI is the preferred imaging modality for detecing and monitoring disease activity in children with JIA . In ordeDespite many efforts to increase the use of MRI to assess the inflamed wrist, imaging the wrist remains a serious challenge in the field of JIA. With the wrist being the most affected location in rheumatoid arthritis, previous studies on (dynamic contrast-enhanced) MRI in the adult wrist provide a good starting point for further evaluation in JIA , 5–20 S. In the The objective of this study was to perform a comprehensive feasibility evaluation of dynamic contrast-enhanced MRI in the wrists of children with JIA using two types of outcome measures: conventional descriptive measures and time-intensity-curve shape analysis including movement registration. Secondly, we aimed to explore the association between enhancement characteristics and clinical disease status.Between March 2012 and July 2013, 32 consecutive children with JIA were prospectively included. Patients were recruited from one of three tertiary outpatient clinics. Institutional Review Board approval was obtained and informed consent was acquired from all parents, as well as from patients older than 12 years of age.Three sets of criteria were used to classify the patients: diagnosis of the JIA subtype was made based on the International League of Associations for Rheumatology criteria , binary classification of disease activity was made based on the Wallace criteria  and a quantification on a continuous scale of disease activity was performed by means of the Juvenile Arthritis Disease Activity Score –28.All patients either had current wrist involvement  or previous wrist involvement . The indication to perform an MRI was always clinically driven. For example, in the case of children with clinically active JIA, the MRI was made to confirm synovial inflammation in the wrist. An example of MRI indication in a clinically inactive patient was to evaluate whether treatment could be downscaled in case of absent synovial inflammation on MRI.Exclusion criteria were a history of intra-articular corticosteroid injection in the wrist within the last 6 months, the need for anesthesia during the MRI examination and general contraindications for MRI.Clinical assessment was performed by one of the experienced pediatric rheumatologists  and consisted of a 67 active joint count defining presence of swelling, pain on motion/tenderness and limited range of motion. Visual analogue scales (100 mm) were used for the physician’s global assessment of overall disease activity, patient’s global assessment of overall well-being and a patient’s pain assessment. The Childhood Health Assessment Questionnaire was used to evaluate patients’ functional abilities . LaboratMRI was performed using an open-bore 1.0-Tesla MRI scanner  and a Sense wrist coil  with the child in a supine position with the arm along the side of the body . No sedaIn-house written codes developed in Matlab  were used for movement registration, drawing of region of interests and extraction of the dynamic contrast-enhanced MRI parameters. We performed non-rigid registration of all dynamic volumes to correct for possible small, residual movements of the wrist. For this movement registration, we adopted a Discrete Cosine Transformation model  with twoA large anatomy-based region of interest was manually drawn on the axial maximal enhancement maps by a radiology trainee (3 years of experience with imaging in JIA), while using the axial post-contrast T1-weighted dynamic contrast-enhanced MR image as a reference. The region of interest was drawn to exclude visible vessels, tendons and the epiphysis of the radius and ulna .Conventional descriptive measures included the median value of maximum enhancement , median maximum initial slope , median initial area under the curve (area under the enhancement curve between start of injection and 90 s after injection) and the enhancing volume .U test was used to assess differences between clinical subgroups. A P-value <0.05 was considered statistically significant. A Spearman’s ρ correlation coefficient was used to assess the association between Juvenile Arthritis Disease Activity Score and dynamic contrast-enhanced MRI measures. Spearman’s ρ was interpreted as follows: <0.19 very weak, 0.20-0.39 weak, 0.40-0.59 moderate, 0.60-0.79 strong and >0.80 very strong. All statistical analyses were performed using SPSS version 20.0 .Descriptive statistics were reported in terms of percentages means, medians, standard deviations and interquartile ranges. Normality plots were used to assess the normal distribution of the data. The Mann-Whitney Thirty-two children with JIA were included . The JIA subtypes were as follows: 6 (19%) persistent oligoarthritis, 4 (13%) extended oligoarthritis, 15 (47%) polyarthritis rheumatoid factor negative, 2 (6%) polyarticular rheumatoid factor positive, 2 (6%) psoriatic arthritis, 2 (6%) enthesitis-related arthritis and 1 (3%) undifferentiated arthritis. The clinical characteristics of the patients divided per subgroup are summarized in Table All children with JIA underwent the dynamic contrast-enhanced MRI without difficulty: the added scan time was negligible in comparison to the complete duration of the MRI. Dynamic contrast-enhanced MRI yielded images with good quality and without appreciable movement artifacts. The motion correction was applied to address small shifts and rotations in the position of the hand between subsequent dynamic scans.P = 0.019). The other conventional descriptive dynamic contrast-enhanced MRI measures did not show any statistical significant differences between children with active and inactive disease  and clinically inactive (median maximum enhancement: 0.179) groups . The children with active disease showed a trend toward higher numbers of time-intensity-curve shapes 3 and 4 compared to the children with inactive disease, but did not reach statistical significance (P = 0.310 and P = 0.166). No difference between active and inactive disease was found with respect to the number of time-intensity-curve shape 5  with the clinical disease activity as reflected by Juvenile Arthritis Disease Activity Score Table .Table 2SIn the current study, we evaluated dynamic contrast-enhanced MRI in children with JIA and wrist involvement, by using both conventional descriptive measures and time-intensity-curve shape analysis. Dynamic contrast-enhanced MRI of the wrist is considered feasible, as the children with JIA could lie in the scanner for the time required for the investigation and image quality of both conventional descriptive measures and time-intensity-curve shape analysis was good. The descriptive dynamic contrast-enhanced MRI parameter maximum enhancement differed significantly between clinically active and inactive JIA disease. No significant larger number of the expected aggressive enhancement patterns (higher relative frequency of time-intensity-curve shapes 3 and 4) in children with clinically active disease could be proven, though a trend toward larger relative frequency of these types was observed.In 2010, the first step towards the use of dynamic contrast-enhanced MRI of children with JIA was made through the assessment of its reliability and construct validity in a pilot study of 12 patients with wrist involvement . The curOn the other hand, one conventional descriptive measures of dynamic contrast-enhanced MRI did prove to be indicative of clinically active disease, as there was a significant difference with the clinically inactive group. The added value of maximum enhancement for distinguishing clinically active versus clinically inactive disease in the JIA wrist is consistent with previous findings in the wrist affected by rheumatoid arthritis and the knee affected by JIA. However, these differences were significant on a group level; no value for an individual difference was shown in this study , 14, 23.The validity of dynamic contrast-enhanced MRI and its potential relevance in JIA was further explored by the correlation with Juvenile Arthritis Disease Activity Score as a clinical measure for disease activity. The only measure reaching a moderate correlation was the enhancing volume, despite the lack of significant difference in enhancing volume between active and inactive disease. On the other hand, correlations between clinical measures and MRI are not expected to be very high, as both entities are considered more complementary than similar.Feasibility of the chosen method for image acquisition with respect to the movement registration and positioning of the arm proved to be good. The movement registration algorithm visibly improved the image quality by correcting for possible movement occurring between scans. The risk of motion artifacts was also reduced by a relatively comfortable position for patients in our center undergoing MRI of the wrist in an open-bore 1.0-T magnet with the arm along the side of the body. Some other MRI systems might suffer from more motion artifacts, due to the required artifact-prone Superman-like position. In this case, the movement registration algorithms would be even more valuable. On the other hand, the arm along the side of the body also has its limitations, especially with respect to higher susceptibility to fold-over artifacts if the arm is not moved far away enough from the body.This most important limitation of this study is the fact that the clinical subgroups were unequally divided. Very few children with clinically inactive disease and previous wrist involvement had a clinical indication for MRI in our patient clinics, which led to a low number of children with inactive disease in our study and, therefore, underpowered statistical analysis. This equal distribution of subgroups deserves attention in future studies. Furthermore, the field strength of 1.0 T, chosen because of the geometrical advantage of the open scanner, could have influenced image quality  and the results. Future dynamic contrast-enhanced MRI studies are preferably performed on machines with a field strength of 1.5 T or higher.This study indicates that dynamic contrast-enhanced MRI is promising as a functional biomarker for children with JIA with wrist involvement and that descriptive parameters of dynamic contrast-enhanced MRI are better associated with clinical disease status of the wrist than time-intensity-curve shape analysis. Currently, the clinician treating children with JIA can use the maximum enhancement and enhancing volume as markers more suggestive of clinically active disease. Further implementation of dynamic contrast-enhanced MRI in clinical practice requires investigation of the possibilities of automated post-processing of the images, as well as the assessment of the added value for the individual child with JIA with respect to treatment and clinical course. This can be achieved by determining whether dynamic contrast-enhanced MRI is, for example, predictive of therapy response or long-term outcome.ESM 1(DOC 44 kb)ESM 2(DOC 74 kb)Below is the link to the electronic supplementary material.


We have discussed a unique presentation of primary diffuse large cell B-cell non-Hodgkin (DLBC NHL) hepatic lymphoma involving the porta hepatis and biliary confluence causing obstructive jaundice with contiguous soft tissue involvement of the right lobe of liver extending up to the right renal cortex. This appears to be the only case in literature where primary hepatic lymphoma has shown contiguous localized intra- and extrahepatic tumor infiltration. A 67-year-old gentleman presented with history of significant loss of appetite and weight in 2 months with associated progressive painless cholestatic jaundice. Physical evaluation revealed normal vitals with pallor, deep icterus, scratch marks over the abdomen, generalized muscle wasting, grade II clubbing and a palpable non-tender liver with a globular, firm mass beneath the liver. He had a total serum bilirubin of 15.9 mg/dL and direct bilirubin of 9.24 mg/dL. His liver enzymes were moderately elevated with raised serum creatinine and dyselectrolytemia. Serology for enterohepatic viruses was negative. Contrast-enhanced magnetic resonance imaging (CEMRI) showed poorly enhancing multiple soft tissue masses in both lobes of liver with the largest mass involving, biliary confluence and porta hepatis causing right bile duct and portal vein encasement. The mass occupied the posterior right lobe and extended to the inferior surface of liver with contiguous invasion of the right renal upper pole cortex. The mass was associated with a retracted liver capsule in the involved segments and delayed enhancement, mimicking a cholangiocarcinoma. Tissue biopsy revealed hepatic DLBC type NHL and patient was subsequently treated with a CHOP-R (cyclophosphamide-doxorubicin-vincristine-prednisolone/rituximab) regimen, on which he has shown non-progressive disease at 1-year follow-up. DLBC NHL of the liver is a very rare tumor with propensity for isolated involvement of the liver and minimal extrahepatic spread. This case shows many interesting features such as obstructive jaundice for 2 months, porta hepatis involvement and tumor infiltration up to the right renal parenchyma. We have illustrated various imaging findings which should be considered when evaluating such a lesion to help differentiate it from cholangiocarcinoma. The literature is extensively reviewed. The case demonstrates relevant diagnostic parameters for physicians, radiologists and oncologists who are likely to encounter patients with tumor-induced obstructive jaundice in their daily practice. Lymphoma is a systemic lymphoproliferative malignancy and is classified into the Hodgkin and non-Hodgkin lymphoma (NHL) subtypes. NHL originates from nodal and extra nodal sites. Primary hepatic lymphoma (PHL) is a very rare subgroup of extra-nodal NHL because of a natural lack of abundant lymphoid tissue in the liver. It is seen in less than 1% of NHL . PHL wasThe diagnostic criterion for PHL is a lesion confined to the liver without spleen, bone marrow or hematological involvement and lack of superficial lymphadenopathy at the time of presentation as well as up to the next 6 months . The criDue to the stringent and well-defined diagnostic guidelines of this group of lymphomas, only few hundred isolated case reports have been described in literature so far. Most of these tumors present as insidious, asymptomatic lesions which are rarely suspected at the time of evaluation and are a histological surprise . The speThe etio-pathogenesis of PHL is unclear; however, few associated factors have been suggested. These include: prior infection with hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), cirrhosis due to primary biliary or secondary to viral infections, auto-immune diseases and immunosuppressive therapy , 9. It hAlthough there are few case reports of surgically treated solitary focal liver lesions in PHL, the universally accepted non-surgical treatment consists of cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab (CHOP-R) regimen, which is the mainstay of therapy for PHL patients.We present a unique case of NHL with obstructive jaundice involving the liver as the primary site. We believe that the soft tissue extension of the tumor to the right kidney along with the other imaging and clinical features makes this a rare case report which is worthy of attention to all physicians and clinicians who observe similar cases in their daily practice.A 67-year-old gentleman presented with history of significant loss of appetite and weight (approximately 42 pounds) in 2 months with associated progressive painless cholestatic jaundice. He also complained of extreme generalized weakness and fatigue. The patient denied any febrile episodes or bleeding manifestations during this period. There was no history of abdominal pain, distension or fullness. No symptoms of altered sensorium leg and face swelling or decreased urine output were seen. Patient denied chronic, addictive substance abuse or use of any indigenous medication during and prior to his illness period. The patient had never undergone surgery or blood transfusions in the past. Physical evaluation revealed the patient was conscious, oriented and afebrile with normal vitals. Pallor was evident and the patient was deeply icteric with evidence of scratch marks over the abdomen. There was generalized muscle wasting and loss of subcutaneous fat over the face and hollowing of the temporal regions. There was presence of grade II clubbing. No evidence of cyanosis or peripheral edema was seen. No peripheral, mediastinal or abdominal adenopathy was found. The abdomen was non-distended without visible venous prominences or visible pulsations. Palpation revealed a firm, rounded, smooth non-tender liver palpable up to 6 cm below the right costal margin. A large globular non-tender firm mass of about 8 cm was palpable beneath the liver. There was no splenomegaly or free fluid. The rest of the systemic examination was normal and non-contributory. His laboratory investigations are shown in Serology for hepatitis A, E, B, and C viruses as well as for HIV was negative. Tumor markers such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) were normal. Serum CA19-9 was elevated. Contrast-enhanced magnetic resonance imaging (CEMRI) using hepatocyte specific contrast gadobenate dimeglumine (gadolinium BOPTA) was performed at our institute which revealed the presence of multiple T1-hypointense and T2-hyperintense intrahepatic (entire right lobe and segment-IV of the left lobe) lesions showing restriction on diffusion weighted images . On contFig. 7-Differential diagnosis on imaging as well as on pathology which was ruled out at tissue biopsy is as follows: 1) hepatocellular carcinoma; 2) cholangiocarcinoma; 3) metastatic carcinoma; 4) metastatic neuroendocrine carcinoma; 5) malignant lymphoma .He was subsequently treated with a CHOP-R regimen, on which he is doing well at 1-year follow-up.PHL is defined as tumor confined to the liver with no evidence of lymphomatous or hematological involvement of other organs in the body. It is rare to find NHL presenting with obstructive jaundice. The patient in our study is of interest because of the rarity of combination of PHL presenting as malignant biliary obstruction. This particular tumor combined multiple interesting features such as multiple hepatic lesions, involvement of porta hepatis, portal vein encasement and biliary obstruction. However the most noteworthy feature that was confounding in its diagnosis was, the fact that the tumor extended to the right renal parenchyma directly from the right lobe of liver crossing the hepatic capsule and Morrison’s pouch fat. This is probably the first case in literature which exhibited such findings. Literature review of published case reports of DLBCL of the liver reveals that presentation of these tumors is an amalgam of various entities . The medLymphomatous liver lesions may be incidentally diagnosed on imaging of clinically asymptomatic patients or on evaluation of terminally sick and morbid cases of fulminant hepatic failure . AbdominOur report extends the range of clinical manifestations from solitary, multiple or diffuse lesions to a locally infiltrating tumor extending beyond the liver capsule. Recognition of such an entity is important for the sake of both diagnosis and management of PHL presenting as obstructive jaundice with extent beyond the liver. Prognosis remains favorable in appropriately treated cases.DLBC PHL of the liver is a rare diagnosis on imaging. This case illustrates the importance of imaging and histopathology correlation in patients presenting with malignant obstructive jaundice. The above case has demonstrated a unique presentation of PHL with biliary, portal vein, perihepatic fat planes involvement as well as contiguous extension of tumor tissue into the right kidney. This report will help clinicians, radiologists, oncologists and pathologists to consider this rare tumor as a differential diagnosis for cholangiocarcinoma as well as a possible cause for malignant obstructive jaundice which can be effectively treated with non-surgical means, offering a favorable prognosis.


Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is an effective and safe technique associated with a very low complication rate for the sampling of lymph nodes in those presenting with mediastinal and hilar adenopathy.” We report a rare case describing the development of pneumomediastinum following EBUS-TBNA in a young patient with mediastinal lymphadenopathy secondary to non-Hodgkin’s lymphoma. Conservative treatment led to spontaneous resolution of the pneumomediastinum. Pneumomediastinum is a rare but possible complication of EBUS-TBNA. Careful follow-up can reduce its severity and the associated morbidity. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is an effective technique for the sampling of mediastinal and hilar adenopathy ,2. MoreoIn a nationwide survey performed by the Japan Society for Respiratory Endoscopy, pneumothorax was documented in 2 patients (0.03%), 1 of whom required tube drainage –8. We reA 42-year-old woman presented to the outpatient Pneumology Clinic with a complaint of a persistent cough without fever over a month and the onset of chest pain a week prior to presentation. A chest X-ray revealed a left-sided perihilar opacity . She repThe clinical presentation of pneumomediastinum can occasionally be misdiagnosed or completely missed because of its vague symptoms –11. MostBed rest and conservative management such as the prophylactic use of analgesics and antibiotics in addition to limitation of oral intake are indicated in patients presenting with pneumomediastinum ,12.Management of pneumomediastinum and pneumopericardium depends upon the clinical severity and underlying etiology of these conditions. Most patients respond adequately to the conservative management strategy outlined above. In rare instances, surgery may be required to treat pneumomediastinum that occurs secondary to severe tracheobronchial disruption . ComplicWe have described a rare complication of EBUS-TBNA. Our patient was known to experience chest pain prior to the bronchoscopy; however, the sudden regression in her chest pain noted after the bronchoscopy led to an underestimation of the possible procedural complication, although the patient’s pneumomediastinum resolved with supportive care. Although rare, clinicians must remain mindful of the complications associated with EBUS-TBNA, and careful follow-up is warranted to reduce the associated morbidity.


Regular use of inhaled corticosteroids (ICS) in patients with obstructive lung diseases has been associated with a higher risk of pneumonia, particularly in COPD. The risk of pneumonia has not been previously evaluated in relation to ICS particle size and dose used.Historical cohort, UK database study of 23,013 patients with obstructive lung disease aged 12–80 years prescribed extra-fine or fine-particle ICS. The endpoints assessed during the outcome year were diagnosis of pneumonia, acute exacerbations and acute respiratory events in relation to ICS dose. To determine the association between ICS particle size, dose and risk of pneumonia in unmatched and matched treatment groups, logistic and conditional logistic regression models were used.14788 patients were stepped-up to fine-particle ICS and 8225 to extra-fine ICS. On unmatched analysis, patients stepping-up to extra-fine ICS were significantly less likely to be coded for pneumonia ; experience acute exacerbations ; and acute respiratory events  compared with patients stepping-up to fine-particle ICS. Patients prescribed daily ICS doses in excess of 700 mcg  had a significantly higher risk of pneumonia  compared with patients prescribed lower doses, irrespective of particle size.These findings suggest that patients with obstructive lung disease on extra-fine particle ICS have a lower risk of pneumonia than those on fine-particle ICS, with those receiving higher ICS doses being at a greater risk. Inhaled corticosteroids (ICS) are widely used in high doses in the management of obstructive lung diseases such as asthma and chroIndeed, regular use of ICS has been linked to several systemic effects, includinWhile most ICS are fine particles with a mass median aerodynamic diameter (MMAD) of 2–4 microns, some extra-fine particle ICS formulations have been produced with a particle MMAD of 1.1 microns which improves airway deposition.–29 Two sWe therefore aimed to compare the risk of pneumonia and other adverse respiratory consequences between fine-particle ICS and extra-fine particle ICS in relation to their dose in patients with obstructive lung disease.www.cprd.com) and the UK Optimum Patient Care Research Database . The CPRD is a governmental, not-for-profit research service, jointly funded by the NHS National Institute for Health Research (NIHR) and the Medicines and Healthcare products Regulatory Agency, a part of the Department of Health. All access and use of anonymised data via the CPRD are carefully controlled under UK and European law and the rules and regulations operating in the NHS. OPCRD has been reviewed and ethically approved by the NHS Health Research Authority to hold and process anonymised data as part of their service delivery (Research Ethics Committee reference: 15/EM/0150). The authors had no access to patient identifying information as part of this study and no informed consent was necessary as the data were anonymised.A historical cohort study was conducted in patients with obstructive lung disease in the UK consisting of a baseline and outcome period. The baseline period (for patient characterisation) was the one-year prior to the index prescription date, at which point asthma or COPD patients had their ICS therapy stepped-up (≥50% dose increase) as either extra-fine particle ICS (efBDP or efCIC) or fine-particle ICS (fluticasone propionate (FP) or BDP). Budesonide was not used in this investigation given the lack of or conflicting evidence that budesonide is associated with increased risk of pneumonia.,24,41,42The study protocol was registered with the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance . Formal ethics and research management approval of the study protocol was obtained from the Anonymous Data Ethics Protocols and Transparency (ADEPT) committee and the Independent Scientific Advisory committee (ADEPTZZ015), which verify the scientific and ethical soundness of all research using OPCRD and CPRD data, respectively. The OPCRD has been approved by the Trent Multi Centre Research Ethics Committee for use in clinical research.Aged: 12–80 years at the Index Prescription DateEvidence of obstructive lung disease treatment defined as at least 2 prescriptions (either as fine-particle ICS or extra-fine particle ICS) for respiratory therapy at baseline Continuation of therapy, defined as ≥2 respiratory prescriptions during the outcome year (i.e. ≥1 in addition to that prescribed at the Index Date)Two years of continuous practice data comprising 1 year baseline data and 1 year of outcome data (up-to-standard data for CPRD patients)Patients with any other chronic respiratory disease, at any time were excluded from the study.Diagnosis of pneumonia: (i) unconfirmed i.e. all unique patients with codes for pneumonia and, (ii) confirmed by chest radiograph or resulting in hospitalisation within one month of pneumonia diagnosis.exacerbation was defined as a course of oral corticosteroids (OCS), hospital admission, or emergency department attendance for asthma during the outcome year. For patients with COPD, exacerbations were defined as an acute course of OCS, antibiotics for a lower respiratory tract infection, or a recorded hospitalisation for COPD.Exacerbation of asthma or COPD: An asthma An acute respiratory event was defined as an occurrence of any of asthma or COPD related hospital admission, or emergency department attendance or acute use of OCS or antibiotics prescribed with lower respiratory consultation (consisting of the following: i) Lower respiratory read codes ; ii) Asthma/COPD review codes excluding any monitoring letter codes; iii) Lung function and/or asthma monitoring; iv) Any additional respiratory examinations, referrals, chest x-rays or events).2 or Mann-Whitney U tests as appropriate.Baseline characteristics of unmatched and matched patients prescribed either fine- or extra-fine particle ICS are described using summary statistics and compared using χThe adjusted odds of patients being coded for pneumonia in the outcome period was compared between unmatched and matched treatment groups using logistic and conditional logistic regression models. The dichotomous outcome for pneumonia coding/coding with confirmation was used as the dependent variable with treatment and potential confounding factors as explanatory variables . Both unadjusted and adjusted odds ratios (OR) with their respective 95% confidence intervals (CI) are reported. Results were adjusted for average daily ICS dose in the outcome and presented by categorised average daily ICS dose in the outcome period, to investigate the effect of ICS dose on developing pneumonia.For the same population, two definitions of exacerbation of obstructive lung disease (asthma exacerbation and acute respiratory event) were used to compare respiratory outcomes between treatment groups, as indicators of clinical effectiveness. Event rates were compared between treatment groups using Poisson regression models. Exacerbation rates were the dependent variable, with treatment and potential confounding factors as explanatory variables  and categorised average daily ICS dose). The adjusted rate ratios (RR) with 95% confidence intervals are reported.All analyses were carried out using IBM SPSS Statistics , SAS software  and Microsoft Office Excel 2013 . Statistically significant results are defined as p<0.05 and trends as 0.05 ≤ p<0.10.Results are reported in accordance with STROBE guidelines for reporting cohort studies.The results for unmatched groups are presented in the main paper and for matched groups in Significant differences were observed between patients who received extra-fine versus fine-particle ICS in the demographics and baseline characteristics, as shown in Patients stepping-up their ICS therapy to extra-fine particle ICS were significantly less likely to be coded for pneumonia  compared to those stepping-up to fine-particle ICS, having adjusted for confounders ; Fig 1.Furthermore, patients stepping-up to extra-fine particle ICS were significantly less likely to experience acute exacerbations ; p<0.001)  and acutAn analysis by average daily ICS dose suggested patients receiving daily ICS doses in excess of 700 mcg  were significantly more likely ; p<0.001) ; Fig 2 tResults for analysis of 6636 uniquely matched patient pairs stepping-up to extra-fine particle ICS compared to those stepping-up to fine-particle ICS are presented in the supporting information. Matched patients stepping-up to extra-fine particle ICS were also significantly less likely to experience acute exacerbations ; p<0.001) (Our data show that patients with obstructive lung disease using fine-particle ICS (FP or BDP) stepping-up their ICS therapy to extra-fine particle ICS (efBDP or efCIC) are significantly less likely to have pneumonia in unmatched patients with a trend for lower incidence in matched patients after adjusting for COPD diagnosis. Furthermore, patients on extra-fine ICS are significantly less likely to experience adverse respiratory outcomes compared to patients stepping-up to fine-particle ICS on both unmatched and matched analysis. Patients on daily ICS doses in excess of 700 mcg  have a significantly increased risk of pneumonia compared with patients on lower doses, irrespective of particle size. To the best of our knowledge, this is the first study to compare the risk of pneumonia between extra-fine and fine-particle ICS in patients with obstructive lung disease in relation to ICS dose.Our findings are particularly pertinent in patients with chronic airway colonisation by pathogenic bacteria, which has been identified as a risk factor for COPD disease exacerbations. AdditionStreptococcus pneumoniae, the most common cause of community acquired pneumonia in patients with COPD.[Streptococcus pneumoniae compared to children not receiving ICS,[The pathophysiological mechanisms that contribute to an increased susceptibility to pneumonia in patients treated with ICS are unclear. In murine models, ICS have been shown to significantly increase alveolar macrophage efferocytosis , thereby reducing their ability to combat microbes, including ith COPD. A recentving ICS, which maving ICS,,22,24,41ving ICS, Although2-agonists (LABA) and/or a LABA and long-acting muscarinic antagonists, for patients with severe or very severe airflow limitation and/or two or more exacerbations per year.[ICS are reported to be overused in the treatment of COPD,46–48 coper year.–51 Furthper year.Similarly in patients with asthma, when low-moderate doses of ICS fail to achieve symptom control, ICS/LABA combination therapy has been shown to improve symptom control, reduce acute exacerbations and improve lung function.,4,53,54 a priori that the data would not be adequately powered for a subgroup analysis of the individual cohorts. However, the different pathophysiology, inflammatory profile and response to treatment in patients with asthma and COPD could partly explain the statistical insignificance of the primary endpoint after adjusting for COPD diagnosis as our cohort was predominantly made up of asthmatics. “Furthermore, although we excluded patients using budesonide from the analysis, an ideal comparative study would be between fine-particle and extra-fine particle fluticasone propionate if and when such a formulation became available”.Our study has strengths and limitations. This study drew on two large primary care databases, the CPRD and OPCRD, allowing us to obtain a large sample of 23,013 patients with obstructive lung disease. There was no difference in the incidence of pneumonia confirmed radiologically or by hospitalisation between COPD and asthma patients, and there is a potential that diagnostic misclassification could have arisen from acute COPD exacerbation episodes, since the patient symptoms may have been similar to those of patients with pneumonia. Nevertheless, as patients receiving extra-fine particle ICS had significantly fewer adverse respiratory outcomes compared with fine-particle ICS, we believe that diagnostic misclassification would not have had a substantial effect on our findings. We clubbed patients with asthma and COPD under one umbrella term of obstructive lung disease, having recognised Moreover, the seasonal and yearly variations seen in the distribution of community acquired pneumonia and our observation period of one-year may have been inadequate to determine a true risk. The extent of the advantage of extra-fine particle ICS over fine-particle ICS may have been overestimated in the unmatched analysis as the matched pairs only showed a trend for a lower risk of pneumonia with extra-fine particle ICS after adjusting for the diagnosis of COPD. The main benefit of a matched cohort analysis is a probable increase in efficiency and the selection of the matching variables is vital. In practIn summary, we have shown important findings relevant to prescribing clinicians in the day-to-day management of patients with obstructive lung disease, where an increased risk of pneumonia and higher rates of adverse respiratory events are more likely with fine-particle ICS compared to extra-fine particle ICS. The benefit-risk ratio is an important measure of safety of drugs and the additional respiratory morbidity observed in our study with higher doses of ICS prescribed more frequently to those on fine-particle ICS favours extra-fine particle ICS for the treatment of obstructive lung diseases, particularly at higher doses. Indeed, our data support guideline directed management that recS1 Fig(JPG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Pseudomonas aeruginosa is found ubiquitously within the environment and is recognised as an opportunistic human pathogen that commonly infects burn wounds and immunocompromised individuals, or patients suffering from the autosomal recessive disorder cystic fibrosis (CF). During chronic infection, P. aeruginosa is thought to form structured aggregates known as biofilms characterised by a self-produced matrix which encases the bacteria, protecting them from antimicrobial attack and the host immune response. In many cases, antibiotics are ineffective at eradicating P. aeruginosa from chronically infected CF airways. Cyclic-di-GMP has been identified as a key regulator of biofilm formation; however, the way in which its effector proteins elicit a change in biofilm formation remains unclear. Identifying regulators of biofilm formation is a key theme of current research and understanding the factors that activate biofilm formation may help to expose potential new drug targets that slow the onset of chronic infection. This minireview outlines the contribution made by exopolysaccharides to biofilm formation, and describes the current understanding of biofilm regulation in P. aeruginosa with a particular focus on CF airway-associated infections.The Gram-negative pathogen  Here, we review our understanding of what controls production of the sticky ‘glue’ that holds bacterial communities together. Pseudomonas aeruginosa is an opportunistic human pathogen that is particularly dreaded by the clinical community. This Gram-negative bacterium has been isolated from burn wounds, and from the respiratory, gastrointestinal and urinary tracts, and wreaks havoc within clinical environments due to its ability to grow on medical equipment such as catheters, stents and ventilators. This creates a high risk of transmission between patients and has afforded P. aeruginosa the dubious accolade of most common nosocomial infection  and flexible metabolism. Whilst most of the time it exists as a relatively harmless microorganism, an infection in immunocompromised individuals or patients receiving immunosuppressive drugs can rapidly become severe enough to result in death of the patient. Treatment options for the infection are also often limited as P. aeruginosa naturally exhibits a high intrinsic resistance to many antimicrobial agents, making it a huge health and economic burden , because it causes irreversible damage to the lung tissue and accelerated lung failure  has revealed the temporal changes which accompany biofilm formation P. aeruginosa mutants that are defective in biofilm formation, an approach that highlighted the importance of Surface Attachment Defective (sad) genes. Several sad genes are required for flagellar synthesis, and functional disruption of these genes blocks biofilm formation at a very early stage; surface attachment. This is because the flagellum is involved in overcoming surface repulsion at the liquid–surface interface, thereby enabling temporary surface attachment during the first stage of biofilm formation. Temporary surface attachment is followed by exploration of the surface via type IV pili-dependent twitching motility  secreted by algA-algD operon. Typically, a membrane-embedded anti-sigma factor, MucA, binds and sequesters the sigma factor AlgU; an interaction stabilised by MucB , of the envelope stress response system in Escherichia coli, AlgW is activated by the cell wall inhibitor D-cycloserine and MucE which respond to envelope stresses such as inhibition of peptidoglycan synthesis, outer membrane perturbation and changes in the expression of outer membrane proteins  unit that make up the Psl pentasaccharide  and its transporter (CdrB). Overexpression of cdrA leads to cell aggregation in liquid culture which has been attributed to CdrA binding to Psl exopolysaccharides. cdrA mutants form weak biofilms in which Psl is no longer tightly associated with the cells suggesting that CdrA cross-links Psl polysaccharides or tethers Psl to the cell membranes to increase stability within the matrix and promote cell aggregation. Strains defective in Psl synthesis also exhibit reduced cell-associated CdrA levels  and digestive enzymes. Several studies have found that surface-bound exopolysaccharides decreased the ability of neutrophils and macrophages to phagocytose the bacteria by blocking the accessibility of bacterial ligands and preventing their recognition -cyclic dimeric guanosine monophosphate (c-di-GMP) is a signalling molecule produced by bacteria to control a whole consort of biological processes. At high intracellular concentrations, c-di-GMP is the key regulator in activating biofilm growth. Intracellular pools of c-di-GMP are modulated by diguanylate cyclases (DGC), which synthesise the molecule, and c-di-GMP phosphodiesterases (PDE), which catabolise it. DGCs are easy to spot because they contain a conserved sequence motif (GGDEF), whereas PDEs contain either an EAL motif or an HD-GYP motif. However, it is very difficult to predict P. aeruginosa c-di-GMP metabolism  and two response regulators (SadA/R). SadS triggers the activation of SadA/R; the EAL domain of SadR has been associated with PDE activity and so may regulate intracellular levels of c-di-GMP. Not only do they promote biofilm formation but the sadARS genes are also important in downregulating the virulence-determining type three secretion system (T3SS) during biofilm formation to maintain a sessile lifestyle  through FleQ  formation. These polar filaments are vital for twitching motility and contribute to the early stages of biofilm formation by promoting attachment to surfaces. FimX is a regulator of T4P which directly binds c-di-GMP, inducing a conformational change which activates its ability to positively regulate T4P assembly at the cell poles Qi et al.. Becauseet al.Pseudomonas species c-di-GMP has been found to regulate secretion systems directly via the ATPase. C-di-GMP represses T3SS while promoting T6SS, consistent with the induction of biofilm formation by high intracellular c-di-GMP  is a global virulence regulator that works in conjunction with its coregulator, cyclic adenosine monophosphate (cAMP), to induce the expression of genes associated with planktonic growth such as flagellum biosynthesis and both T2SS and T3SS. C-di-GMP interferes with this process by reducing the levels of cAMP but the mechanism by which this occurs remains unclear  RsmA in the cell  sequence of pslA is made unavailable through basepairing with an anti-SD sequence, preventing access by the ribosome and abolishing psl gene translation  of LadS activating its kinase activity and ultimately promoting biofilm formation. The DISMED2 domain of LadS closely resembles some carbohydrate-binding modules; however, the presence of an additional helix inhibits this binding but allows for the binding of calcium instead -based systems, las and rhl, the alkyquinolone system, pqs, and the recently identified integrated QS (IQS) system which responds to phosphate limitation. These interlink in a hierarchical manner with the rhl and pqs systems under the control of the las system  signalling molecules. When grown in planktonic cultures, clinical isolates predominately produce rhl signal (BHL) whereas domesticated laboratory strains produce more las signal (OdDHL). However, when the laboratory strain, PAO1, was grown as a biofilm, the QS profile inverted and resembled more the acyl-HSL pattern observed in CF sputum samples. Using acyl-HSL profiles as biomarkers has since provided evidence that P. aeruginosa does in fact exist primarily as a biofilm in the CF lung (Singh et al.QS signals have now been developed as biomarkers for biofilm formation. Clinical isolates have a characteristic ratio of The aforementioned regulatory pathways do not work in isolation and are constantly feeding into one another to create a complex network that fine tunes the bacteria's response to environmental changes. Unfortunately, in most instances the environmental cues remain unknown but it is likely that the bacteria react to the high-stress environment of the lung in order to protect against dehydration, ROS and oxygen limitation (Mulcahy and Lewenza Pseudomonas aeruginosa is one of the most highly studied and clinically significant organisms in relation to biofilm formation (Müsken et al.et al.P. aeruginosa infections showing that targeting Psl is important in the body's own attempts to disperse persistent biofilms (Baker et al.The presence of a heterologous population makes finding antibiotic treatment that is effective on the whole biofilm extremely difficult. For example, cap-forming and stalk-forming subpopulations will often display differential tolerance levels to antimicrobial compounds (Yang Here we have highlighted how c-di-GMP signalling, QS and the Gac/Rsm pathway converge to coordinate the expression of exopolysaccharide production and inversely downregulate the expression of planktonic-associated phenotypes. Understanding the regulatory pathways that underpin the synthesis of matrix components means that more and more regulators can be targeted to prevent biofilm development, not just in CF infections but also in burn wounds and ventilator-associated diseases. The regulatory systems mentioned in this minireview are only the tip of the iceberg for biofilm regulation and so research must continue to uncover the secrets behind this resilient bacterial way of life.EM is supported by a Doctoral Training Grant from the MRC. Work in the laboratory of MW is supported by the BBSRC, NC3Rs and the Rosetrees Trust.Conflict of interest.None declared.


To date, however, little consideration has been given to combining gene-based tests of association for the analysis of pedigree data. We propose a flexible framework for combining any number of family-based rare-variant tests of association into a single summary statistic and for assessing the significance of that statistic. We show that this approach maintains type I error and improves the robustness, to different genetic architectures, of the statistical power of family- and gene-based rare-variant tests through application to simulated phenotype data from Genetic Analysis Workshop 19.Current rare-variant, gene-based tests of association often suffer from a lack of statistical power to detect genotype–phenotype associations as a result of a lack of prior knowledge of genetic disease models combined with limited observations of extremely rare causal variants in population-based samples. The use of pedigree data, in which rare variants are often more highly concentrated than in population-based data, has been proposed as 1 possible method for enhancing power. Methods for combining multiple gene-based tests of association into a single summary  Over the past decade, the rapid decrease in costs for DNA sequence data have made it possible to consider the association of rare single nucleotide variants (SNVs) and complex disease phenotypes. However, the power of any single variant test of genetic association on such rare variants is limited. As such, many gene-based tests of association  have recently been proposed, with the intent to improve statistical power over single marker association tests. Recent insights have classified most of these gene-based tests of association into 2 broad classes: burden  tests and variance component  tests . Burden Despite improvements over single-marker approaches, gene-based tests may still have limited utility in detecting causal rare variants because of an overall lack of power. This lack of power is exacerbated by the fact that optimizing the limited power of gene-based tests of association (by selecting the appropriate burden or variance component test) requires some a priori knowledge of the underlying genetic architecture. In particular, burden tests are most powerful when the variants within a SNV set are mostly causal and have the same causal direction, whereas variance component tests perform optimally in circumstances where the causal direction varies  , even fop values from the gene-based tests of association into one summary p value for the gene, but are frequently limited by not being applicable to family-based data. One exception is the seqMeta package in R [In response, new tests of association have been proposed that simultaneously leverage the strengths of both burden and variance component tests –5. Theseage in R .The use of family/pedigree data has become popular as another means of increasing statistical power to detect causal rare variants. Rare SNVs are more concentrated in affected families, meaning that causal SNVs may aggregate in pedigree data more than in the general population or in a traditional case-control study. In this article, we propose a general method of combining different rare-variant tests for use on family-based data. We then quantify to what extent combining family-based rare-variant tests of association increases power and maintains type I error rate for simulated phenotypes using data from Genetic Analysis Workshop 19 (GAW19).p values across a variety of family-based gene-level rare-variant tests of association into a single summary statistic. Given a set of k distinct family- and gene-based rare-variant tests of association, the method involves the following 5 steps: (a) Generate a vector of test statistics ith family-based rare-variant test of association . (b) Permute the phenotype of interest m times. Find jth permutation . (See next paragraph for consideration of pedigree structure.) (c) Convert each test statistic vector, p values, where m permutations, and S is a univariate statistic calculated by combining jth permutation. In this article we use p value combination options are possible. (e) Compute the significance level of m.Our approach, which extends the approach by Derkach et al  to the cPermuting phenotype values when pedigree structure exists in the samples fails to maintain appropriate type I error rates. To address this limitation, we modify the approach listed in the previous paragraph by first fitting a linear mixed effects model to the desired covariates, with familial correlation as a random effect, and the phenotype of interest as the response, as has been done in related settings . We estiAs a proof-of-concept, we applied the above method to data from GAW19. The data consists of real genotype data, on which blood pressure phenotypes were simulated, for 849 Mexican American individuals across 20 separate pedigrees. We considered the continuous response variable mean arterial pressure (MAP), calculated as (2/3)*(diastolic blood pressure) + (1/3)*(systolic blood pressure). We assessed the relationship between MAP and the 30 genes on chromosome 3 that possess at least 1 casual variant using 1000 permutations per test. Power was then calculated as the proportion of times among the 200 individual phenotype simulations where each gene was found to be significant, using a 0.05 threshold. Type I error was assessed in a similar method on the same 30 genes, but with the trait Q1  as the phenotype of interest.ith variant, ith genotype vector. In particular, 2 different variant weighting systems were considered. The weights suggested by Wu et al. [ith variant, are calculated using a Beta distribution and substantially downweight common variants,  Table .Table 1Tp value procedure, the combined test had power in the same range as more optimal tests. We further note that, depending on the characteristics of the gene being examined, tests featuring either the PDCD6IP, which contains a number of highly causal rare variants, and ZBTB38, which contains several causal common variants .Pairwise correlation between Few current methods allow for a flexible combination of family-based rare-variant tests of association. In response, we propose an adaptable framework for combining any number and type of gene-based rare-variant tests on pedigree data that condition on genotype data. Unlike current existing combination methods, our proposed method does not limit the researcher to combining just 1 family-based burden test with 1 family-based variance-component test, but instead allows for the combination of an arsenal of varied and specialized tests. Our proposed method also offers a distinct advantage over simply running all possible individual tests and choosing the most significant result; our combination approach appropriately controls for multiple testing and for the correlation between tests. When applied to the GAW19 data, the combination method appeared to sufficiently maintain type I error and demonstrated generally robust statistical power, even when applied to a wide variety of genetic architectures, or when low-powered tests were included.p value).The permutation component of our combination approach also poses computational challenges. The process of calculating p values when asymptotic distributions have not been derived is time intensive. The tests evaluated here were applied to only 30 genes in a single simulated data set. We note that if combining tests with known asymptotic distributions, it is straightforward to utilize asymptotically obtained p values from individual tests when creating However, our current approach does have several limitations. In the above proof-of-concept application, the combined test incorporates 8 highly correlated individual tests of association; the additional noise from this correlation diminishes the power of the combined test to detect any meaningful genotype–phenotype associations. Further research is needed to explore what combination of tests minimizes such correlation while also maximizing statistical power. Further work is also necessary to evaluate the benefits and drawbacks of different choices of combined test statistics (eg, alternatives to using the minimum entclass1pt{minimaOur analysis acts as a proof-of-concept for combined tests on family-based rare-variant data. In particular, our analysis suggests that the type I error rate is controlled by the proposed method, while power may be more robust to differing genetic architectures than individual, gene-based rare-variant tests. Further work is needed to explore the trade-off between combining many tests versus combining a smaller set of diverse tests in other data sets/genes to ensure transferability of these findings. Additionally, we need to explore alternatives to permutation strategies to compute statistical significance resulting from the inherent computational limitations of this approach.


Graphene-based materials are elective materials for a number of technologies due to their unique properties. Also, semiconductor nanocrystals have been extensively explored due to their size-dependent properties that make them useful for several applications. By coupling both types of materials, new applications are envisaged that explore the synergistic properties in such hybrid nanostructures. This research reports a general wet chemistry method to prepare graphene oxide (GO) sheets decorated with nanophases of semiconductor metal sulfides. This method allows the in situ growth of metal sulfides onto GO by using metal dialkyldithiocarbamate complexes as single-molecule precursors. In particular, the role of GO as heterogeneous substrate for the growth of semiconductor nanocrystals was investigated by using Raman spectroscopic and imaging methods. The method was further extended to other graphene-based materials, which are easily prepared in a larger scale, such as exfoliated graphite oxide (EGO).  The solid obtained, was thoroughly washed and collected by filtration.The following metal diethyldithiocarbamates were used as single-molecule precursors {M[S, and Pb . All of Exfoliated graphite oxide was produced by the sonochemical exfoliation of graphite in a high boiling point solvent (DMF), in accordance with the literature . In a ty2.All procedures were performed in a well-ventilated fume cupboard. The GO sheets were decorated with metal sulfides by the addition of ethylenediamine (9 mmol) to a dry ethanol suspension of GO  containing the metal precursor (58 μmol). The mixture was then stirred at reflux until a color change of the reaction mixture was clearly observed. The hybrid nanostructures were then collected by centrifugation  using an Eba 20 Hettich centrifuge and thoroughly washed with ethanol. Finally, the powders were dried at room temperature and kept under Nα monochromatic radiation source at 45 kV/40 mA. The samples were prepared by the deposition of aliquots of the ethanolic suspensions in hybrid nanostructures on a silicon holder. XRD patterns of the GO-MS nanocomposites with the internal reference (Si) were recorded similarly. For transmission electron microscopy (TEM) analysis, a drop of the suspension under analysis was placed on a carbon-coated Cu grid and the solvent was left to evaporate at room temperature. TEM was performed using a transmission electron microscope Hitachi H-9000  operating at 300 kV. The high-resolution TEM images were acquired by using a JEOL 2200FS HR-TEM instrument . SEM analysis was performed using a scanning electron microscope Hitachi SU-70  operating at an accelerating voltage of 15 kV. Energy dispersive X-ray spectroscopy (EDX) analysis was performed using Bruker Esprit . Raman spectral imaging was performed in a combined Raman-AFM confocal microscope WITec alpha300 RAS+ . A Nd:YAG laser operating at 532 nm was used as excitation source with the power set at 1 mW. The Raman confocal microscope was calibrated by acquiring the spectrum of a silicon wafer . The Gaussian function was fitted to the Raman band at 521 cm−1, and an error of 0.08 cm−1 was obtained. The intensity values and associated errors of the Raman bands of pristine GO and the nanocomposites were obtained by multi-peaks fitting using the Gaussian function in Origin 8. Raman imaging experiments were performed by raster-scanning the laser beam over the samples and accumulating a full Raman spectrum at each pixel. The WITec software, WITec Project 2.0 , was used to create the Raman images. The Raman images showing a color scale have been created using band integrals, in which the value of the absolute area underneath a band  corresponds to color intensity in the scale, shown in the image at the respective pixels. The Raman spectra of the semiconductor and the GO were used as the basis set in the analysis using the software tool of WITec Project, providing the color-coded combined Raman images.The UV/Vis spectra were recorded using a Jasco V-560 spectrometer . The X-ray power diffraction (XRD) data were collected using a PANanalytical Empyrean X-ray diffractometer  equipped with Cu-KGO-metal sulfide hybrid nanostructures have been successfully produced by in situ thermal decomposition of the respective metal alkyldithiocarbamato complexes. This one-step method is scalable for producing hybrid GO materials with negligible phase segregation and might be extended to other graphene-based materials. The method produces GO materials decorated with metal sulfides, whose absorption in the visible can be tuned by the deposited semiconducting nanophase. We are currently exploring this approach to trigger photon harvesting in water compatible photocatalysts that perform efficiently in the visible spectral region.


The beginning of the 21st century was marked by a number of severe summer floods in Central Europe associated with extreme precipitation . Extratropical storms, known as Vb-cyclones, cause summer extreme precipitation events over Central Europe and can thus lead to such floodings. Vb-cyclones develop over the Mediterranean Sea, which itself strongly warmed during recent decades. Here we investigate the influence of increased Mediterranean Sea surface temperature (SST) on extreme precipitation events in Central Europe. To this end, we carry out atmosphere model simulations forced by average Mediterranean SSTs during 1970–1999 and 2000–2012. Extreme precipitation events occurring on average every 20 summers in the warmer-SST-simulation (2000–2012) amplify along the Vb-cyclone track compared to those in the colder-SST-simulation (1970–1999), on average by 17% in Central Europe. The largest increase is located southeast of maximum precipitation for both simulated heavy events and historical Vb-events. The responsible physical mechanism is increased evaporation from and enhanced atmospheric moisture content over the Mediterranean Sea. The excess in precipitable water is transported from the Mediterranean Sea to Central Europe causing stronger precipitation extremes over that region. Our findings suggest that Mediterranean Sea surface warming amplifies Central European precipitation extremes. Observational records show no clear trend in the intensity of summertime heavy rainfall in Central Europe123567810Flood producing summer precipitation extremes in Central Europe are often associated with Vb-cyclones121316The Mediterranean is itself a hotspot of global warmingSummer precipitation extremes in Central Europe may be amplified due to changes in convective storm dynamics  or changes in cyclone-related precipitation. For instance, there are two major factors that may contribute to the intensification of cyclone-related summer precipitation extremes in Central Europe: changes in cyclone occurrence or pathway (dynamic changes) and changes in the amount of moisture carried by individual cyclones (thermodynamic changes). Yet, confidence in the thermodynamic aspects of climate change is generally higher than in dynamic changes1414warm), we employ the warmer 2000–2012 SST climatology only in the Mediterranean and Black Seas (see warm), the AGCM is forced globally with monthly climatological fields of 2000–2012 SST and SIC. For each experiment we computed a 40-member ensemble of one year. The model is integrated at a relatively high horizontal resolution for a global atmosphere model of T159  and with 31 vertical levels. Forcing conditions for all three experiments were taken from the Hadley Centre Sea Ice and Sea Surface Temperature dataset (HadISST)In this study, by conducting sensitivity experiments with the atmosphere general circulation model (AGCM) ECHAM5We analyse summer precipitation extremes and the associated atmospheric mechanisms in the three model experiments. We define heavy-precipitation events as days where daily precipitation exceeds the 95th percentile of all summer (JJA) days (wet and dry). To have the same sample size for all simulations we individually choose the 95th percentile of all summer days as threshold in each experiment, i.e., in all experiments the 5% of all summer days with the heaviest precipitation are analysed. Differences between the 40-year-long experiments are compared for (i) climatological seasonal summer means over all summer days (wet and dry), and (ii) mean heavy-precipitation events as represented by the mean threshold excess (i.e. the mean of the 5% heaviest precipitation events within 40 summers). We considered the mean threshold excess for comparison, as extreme values do not follow a normal distribution - neither block maxima nor threshold exceedances. Given the large number of excesses, the Central Limit Theorem applies, and we can thus assume a normal sampling distribution for the arithmetic meanWe model daily precipitation extremes based on heavy-precipitation events  with the generalised Pareto (GP) family of distributionsFor cyclone identification and tracking we use the numerical algorithm of the P.P. Shirshov institute30Pfahl and Wernli, 2012Composites of relevant variables are built on days of area-aggregated heavy-precipitation events over the study region located at 15–22°E, 46–51°N  is 2 mm/day. The Central European mean climatological summer cyclone track density is 27 cyclones per summer in both experiments  is simulated in the Carpathian Basin; the largest decrease  in northeastern Germany. Note that the highest increase is not co-located with the region of maximum precipitation on heavy events . This is clearly indicated by track densities of cyclones that passed through the study region on event days . The western Mediterranean Sea is identified as the major moisture source region; this is also true for the additional moisture in Medwarm which is supported by the change in evaporation . The largest increase is located southeast of the precipitation maximum for both simulated heavy events and historical Vb-events. For intensified extreme precipitation changes in transients passing to Central Europe, moisture recycling or the dominant synoptic pattern are not required. The heavier precipitation events are likely related to thermodynamic changes: the stronger diabatic heating source along with increased water vapor content, intensifies moisture transport from the western Mediterranean Sea to Central Europe during heavy-precipitation events. These findings are in line with previous studies on thermodynamic changes21Our atmosphere-only experiments do not represent atmosphere-ocean feedbacks. We rather investigate the atmospheric response to assumed externally forced long-term SST changes. Our results are subject to the chosen atmospheric general circulation model realistically simulating the underlying processes. Yet, the major effect of increasing SSTs was thermodynamic, for which confidence is generally high compared to dynamical changesAmplified Mediterranean SSTs are both forced anthropogenically14How to cite this article: Volosciuk, C. et al. Rising Mediterranean Sea Surface Temperatures Amplify Extreme Summer Precipitation in Central Europe. Sci. Rep. 6, 32450; doi: 10.1038/srep32450 (2016).


The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis of published GWA studies. We propose that dysregulation of monocyte adaptation to the environment of the gastrointestinal mucosa is the key process leading to inflammatory bowel disease. Macrophages are immune cells that form the first line of defense against pathogens, but also mediate tissue damage in inflammatory disease. Macrophages initiate inflammation by recognising and responding to components of bacterial cells. Macrophages of the wall of the gut are constantly replenished from the blood. Upon entering the intestine, newly-arrived cells modulate their response to stimuli derived from the bacteria in the wall of the gut. This process fails in chronic inflammatory bowel diseases (IBD). Both the major forms of IBD, Crohn’s disease and ulcerative colitis, run in families. The inheritance is complex, involving more than 200 different regions of the genome. We hypothesised that the genetic risk of IBD is associated specifically with altered regulation of genes that control the development of macrophages. In this study, we used the comprehensive transcriptome dataset produced by the FANTOM5 consortium to identify the sets of promoters and enhancers that are involved in adaptation of macrophages to the gut wall, their response to bacterial stimuli, and how their functions are integrated. A reanalysis of published genome-wide association data based upon regulated genes in monocytes as candidates strongly supports the view that susceptibility to IBD arises from a primary defect in macrophage differentiation. Inflammatory bowel disease (IBD) comprises a group of complex syndromes that arise from a dysfunctional interaction between the microbiota of the intestinal lumen and the immune system . Loci as+ monocytes for 4–5 days in CSF1 , rheumatoid arthritis [http://plaza.umin.ac.jp/~yokada/datasource/software.htm] [http://csg.sph.umich.edu/abecasis/ public/lipids2013/] [http://portals.broadinstitute.org/collaboration/giant/index.php/GIANT_consortium_data_files] [To assess the association of these candidates with IBD susceptibility, Crohn’s disease summary p-values were obtained from the International Inflammatory Bowel Disease Genetics Consortium  were counted in 1000 equally-sized bins above and below all TSS in the input set (observed counts), and compared to the absolute count of all variants genotyped in the same study in the same distance bins from all TSS in the input set (expected counts under the null hypothesis that no enrichment exists). For Crohn's disease, ulcerative colitis and the mixed phenotype of inflammatory bowel disease, linkage disequilibrium score regression analyses reveal strong evidence of polygenicity, supporting our decision to draw inferences from variants that do not meet stringent criteria for genome-wide significance. These analyses were conducted by Bulik-Sullivan et al  using thS1 Fig(PNG)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


The lethality of endometrioid endometrial cancer (EEC) is primarily attributable to advanced-stage diseases. We sought to develop a biomarker model that predicts EEC surgical stage at the time of clinical diagnosis.P < 0.0001) and in the validation cohort (P = 0.0003). Even among grade 1 or 2 tumors, PSES was significantly higher in advanced than in early stage tumors in both the TCGA (P = 0.005) and MD Anderson Cancer Center (MDACC) (P = 0.006) cohorts. Patients with positive PSES score had significantly shorter progression-free survival than those with negative PSES in the TCGA  and validation  cohorts. The ErbB signaling pathway was most significantly enriched in the PSES proteins and downregulated in advanced stage tumors.PSES was significantly correlated with surgical stage in the TCGA cohort  scheme comprising 6 proteins (3 of them phosphorylated) for surgical stage prediction. We validated and evaluated its diagnostic potential in an independent cohort of 184 EEC cases obtained at MDACC using receiver operating characteristic curve analyses. Kaplan-Meier survival analysis was used to examine the association of PSES score with patient outcome, and Ingenuity pathway analysis was used to identify relevant signaling pathways. Two-sided statistical tests were used.PSES may provide clinically useful prediction of high-risk tumors and offer new insights into tumor biology in EEC. Endometrial carcinoma is the most common gynecologic malignancy; over 10,470 deaths from uterine corpus cancer were expected in the United States in 2016 . PatientTP53 mutation,  and finds a local minimum of the custom-defined function described in Fun(x).hi(x)PSES and lo(x)PSES are the PSES scores for advanced and early stage tumors respectively and depend upon the weighting factor, x. ROC denotes the ROC curve analysis of these two scores; the corresponding value of the area under the curve (AUC) for each x was then calculated. Minimizing Fun(x) is equivalent to maximizing the predictive performance assessed by receiver operating characteristics (ROC) curve analysis. The x hat represents the optimized weighting factor vector that gives rise to a maximum AUC value. The ROC curves evaluated via PSES scores were calculated as examples at the initialization and at the end of optimization , SPSS version 18 , and GraphPad Prism, version 6 .Survival analyses were performed using the Kaplan-Meier method, and the difference in survival was examined with the use of log-rank tests. Overall survival (OS) was defined as the interval from the date of initial surgical resection to the date of last known contact (censored) or death. Progression-free survival (PFS) was defined as the interval from the date of initial surgical resection to the date of progression, recurrence, or last known contact (censored). Statistical significance was defined as


Feedback, particularly real-time feedback, is critical to resident education. The emergency medicine (EM) milestones were developed in 2012 to enhance resident assessment, and many programs use them to provide focused resident feedback. The purpose of this study was to evaluate EM residents’ level of interest in receiving real-time feedback on each of the 23 competencies/sub-competencies.This was a multicenter cross-sectional study of EM residents. We surveyed participants on their level of interest in receiving real-time on-shift feedback on each of the 23 competencies/sub-competencies. Anonymous paper or computerized surveys were distributed to residents at three four-year training programs and three three-year training programs with a total of 223 resident respondents. Residents rated their level of interest in each milestone on a six-point Likert-type response scale. We calculated average level of interest for each of the 23 sub-competencies, for all 223 respondents and separately by postgraduate year (PGY) levels of training. One-way analyses of variance were performed to determine if there were differences in ratings by level of training.The overall survey response rate across all institutions was 82%. Emergency stabilization had the highest mean rating (5.47/6), while technology had the lowest rating (3.24/6). However, we observed no differences between levels of training on any of the 23 competencies/sub-competencies.Residents seem to ascribe much more value in receiving feedback on domains involving high-risk, challenging procedural skills as compared to low-risk technical and communication skills. Further studies are necessary to determine whether residents’ perceived importance of competencies/sub-competencies needs to be considered when developing an assessment or feedback program based on these 23 EM competencies/sub-competencies. Real-time feedback during a clinical shift in the emergency department is an important component of a resident physician’s medical education and can have a profound impact on clinical practice.6The Accreditation Council for Graduate Medical Education (ACGME) introduced the Next Accreditation System (NAS) in 2012, which includes 23 emergency medicine (EM) competency / sub-competency domains, each comprised of five levels of specific developmental milestones. This model is the main assessment framework of the NAS. Physicians are expected to progress through the milestone levels of each competency / sub-competency from novice intern to expert.10Various EM studies have revealed widespread dissatisfaction with feedback despite the employment of a wide variety of feedback methods. Most studies on feedback involve attending- or program leader-initiated feedback. Few have explored the theme of learner-initiated feedback. The objective of this research project was to evaluate EM residents’ level of interest in receiving real-time feedback on each of the 23 competencies/sub-competencies. Identifying the areas of most importance to learners may be the first step in helping mitigate issues with poor feedback and giving learners more autonomy over desired feedback.http://www.surveymonkey.com) were distributed to residents of all postgraduate year (PGY) levels at each of the six training programs with a total of 272 possible resident respondents. The project was deemed exempt by the IRB at the Icahn School of Medicine at Mount Sinai followed by review at the remaining institutions.This was a multicenter cross-sectional study of EM residents at six ACGME-accredited academic EM residency programs in the United States. The programs span various regions of the country with three three-year and three four-year residency programs in both urban and suburban settings . ParticiWe surveyed all residents at the six academic EM residency programs regarding their interest levels in receiving feedback by the EM attending during a clinical shift on specific topic areas covering the 23 ACGME EM competencies/sub-competencies. Surveys were distributed at each institution during the middle of the academic calendar year via paper survey and then subsequently via email to capture residents who were not able to complete paper forms. Completion of the survey was considered consent for the study. Study participation was anonymous and voluntary. We provided residents the survey questionnaire  along wiFor content validity, the survey was designed to include all 23 competencies/sub-competencies. To optimize content and internal structure evidence, we created the survey instrument using an iterative editing approach. This included extensive testing among the authors for item generation, survey functionality, matching of item content to the construct, optimal item phrasing, and overall quality control. For response process validity, the survey was piloted by six EM attending physicians and six EM senior resident physicians and subsequently revised.Residents rated their level of interest in receiving on-shift feedback on each competency/sub-competency using a six-point Likert-type response scale . We calculated average levels of interest for each of the 23 competencies/sub-competencies for all respondents and by PGY level of training. A one-way analysis of variance (ANOVA) was conducted to determine whether differences in desire for feedback existed by level of training (PGY level). To control for Type-1 error rates from multiple comparisons, we adjusted the p-value for significance using the Bonferroni correction suggested by Bland, 1995 (p=.05/23 tests= .002).12The overall survey response rate was 82% (223/272). Return rates and residency characteristics are detailed in One-way ANOVA analyses  showed nThe EM Milestones project, developed by the ACGME and the American Board of Emergency Medicine, provides residency programs with descriptive, objective criteria by which to assess a resident’s progress throughout his or her training. While program directors and academic faculty in residency programs are familiar with the milestone sub-competencies, it is less clear if residents have similar investment in the tools being used to evaluate them. Some residents may have little to no knowledge about each of the individual competencies/sub-competencies and the criteria used to differentiate various levels of performance on the milestones scale. Residents may also not internalize feedback on competencies/sub-competencies for which they feel are not relevant to them at a given time. This study aimed to assess EM residents’ interest in receiving real-time feedback on each of the 23 different EM competencies/sub-competencies.Of the 23 competencies/sub-competencies, residents were most interested in receiving feedback on three: emergency stabilization, airway management, and medical knowledge. Compared to the other milestones, these seem to reflect the core values of the practice of EM – complicated skill sets that are high reward, if done well, and have significant impact on patient outcomes. Of these, emergency stabilization and medical knowledge encompass broad content areas covered during residency education.There was one outlier competency on which residents were least interested in receiving feedback: technology and electronic health records. This competency had the lowest average interest rating at 3.24 out of 6, reflecting mild interest in receiving feedback. Possible explanations for why this milestone was least interesting to residents include lack of understanding of its importance in their future career, lack of perceived relevance to direct patient outcome, difficulty in receiving feedback on this work, or even perceived adequacy of prior or current feedback on this competency.All other competencies/sub-competencies received ratings between 3.6–4.9, reflecting significant resident interest in receiving feedback on these topics. By rating all of the competencies/sub-competencies as at least mildly interesting regarding feedback, residents are validating the idea that the competencies/sub-competencies accurately represent relevant learning objectives throughout residency that are perceived as applicable to their future practice. There were no statistically significant differences between residents based on PGY level regarding their interest in milestone-based feedback, suggesting that feedback on any of the competencies/sub-competencies would be appreciated at any learner level.Prior work suggests that a trainee’s prior experiences, confidence level, fear of appearing incompetent, and biases in cognitive reasoning processes can affect their responsiveness to feedback.A limitation of this study is the variability in response rates across the participating institutions. The lowest survey response rate at a site was 69% while the site with the highest response rate was 93%. However, such a diverse subject population is important for allowing generalizability of aggregate resident survey responses across the larger group of EM trainees across the country.To obtain the highest possible response rate, some residents were given a paper survey while others participated in the online survey. The different vehicles by which certain residents responded may have affected the responses given.Providing effective feedback to residents is essential to their education and professional growth. Residents frequently report discontent with the feedback they receive, and thus a better understanding of feedback and residents’ preferences regarding feedback may allow attending physicians to provide more useful feedback. We observed no differences between resident levels of training, suggesting that preference for feedback is unrelated to PGY level. Future areas of research in this domain include elucidating whether feedback is more effective if it involves a sub-competency of particular interest to the resident, and if sub-competencies deemed “less interesting” require particular attention to reinforce their importance in a resident physician’s career.


Isoflavones are polyphenols with estrogenic activity found mainly in soy and soy-derived products that need to be metabolised in the intestine by the gut bacteria to be fully active. There is little knowledge about isoflavone bioconversion and equol production in the human intestine. In this work, we developed an in vitro anaerobic culture model based on faecal slurries to assess the impact of isoflavone supplementation on the overall intestinal bacterial composition changes and associated metabolic transformations.Collinsella, Faecalibacterium and members of the Clostridium clusters IV and XIVa. In addition, the concentration of short-chain fatty acids (SCFAs) detected in the isoflavone-containing faecal cultures was higher in those inoculated with faecal slurries from equol-producing women.In the faecal anaerobic batch cultures of this study bioconversion of isoflavones into equol was possible, suggesting the presence of viable equol-producing bacterial taxa within the faeces of menopausal women with an equol producer phenotype. The application of high-throughput DNA sequencing of 16S rRNA gene amplicons revealed the composition of the faecal cultures to be modified by the addition of isoflavones, with enrichment of some bacterial gut members associated with the metabolism of phenolics and/or equol production, such as This study constitutes the first step in the development of a faecal culturing system with isoflavones that would further allow the selection and isolation of intestinal bacterial types able to metabolize these compounds and produce equol in vitro. Although limited by the low number of faecal cultures analysed and the inter-individual bacterial diversity, the in vitro results obtained in this work tend to indicate that soy isoflavones might provide an alternative energy source for the increase of equol-producing taxa and enhancement of SCFAs production. SCFAs and equol are both considered pivotal bacterial metabolites in the triggering of intestinal health-related beneficial effects.The online version of this article (doi:10.1186/s12866-017-1001-y) contains supplementary material, which is available to authorized users. Isoflavones are polyphenols with estrogenic activity found at relatively high concentration in soy and soy-derived products. Epidemiological studies suggest high intakes of isoflavones to be associated with fewer and less intense menopause symptoms, and a reduced incidence of hormone-dependent and aging-associated diseases such as osteoporosis, cardiovascular diseases and cancer . AlthougCoriobacteriaceae  were measured using a UHPLC procedure based on a method for equol determination in urine . Brieflyv/v) with 1 mg/ml of 2-ethyl butyric acid (Sigma-Aldrich) dissolved in methanol as an internal standard. A chromatographic system composed of 6890 N GC apparatus (Agilent Technologies) connected to a flame ionization detector was used to identify and quantify the SCFAs as described elsewhere . (PPTX 164 kb)Effect of isoflavones on dominant bacterial populations determined by DGGE. PCR-DGGE profiles of the primary faecal cultures from equol-producing women grown in modified medium for colonic bacteria supplemented Additional file 3:p value <0.05) increases (grey) and decreases in their relative abundances (% sequences) when comparing primary faecal cultures in medium with and without isoflavones. (DOCX 16 kb)Effect of isoflavones in microbial abundance. Families and genera showing significant  in their relative abundances (% sequences) in medium with isoflavones when comparing primary cultures from non-producer and equol producer women. (DOCX 16 kb)Differences in microbial genera associated with equol production in primary faecal cultures. Genera showing significant increases  in their relative abundances (% sequences) in medium with isoflavones when comparing secondary cultures that rendered equol production with those that did not. (DOCX 17 kb)Differences in microbial genera associated with equol production in secondary faecal cultures. Genera showing significant increases  in their relative abundances (%) in primary faecal cultures in mMCBISO as compared to that in mMCB. (DOCX 17 kb)Effect of isoflavones in microbial species abundance. Identification of OTUs (3% distance level) showing significant increases (