Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography.

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Low-dose ipilimumab plus nivolumab combined with IL-2 and hyperthermia in cancer patients with advanced disease: exploratory findings of a case series of 131 stage IV cancers - a retrospective study of a single institution.

The 3-year overall survival (OS) rate of patients with previously treated or untreated stage III or IV melanoma has by now reached 63% using ipilimumab and nivolumab therapy. However, immune-related adverse events (irAEs) of grade 3 or 4 occurred in 59% of patients leading to discontinuation of therapy in 24.5% of patients and one death. Therapy with checkpoint inhibitors could be safer and more effective in combination with hyperthermia and fever inducing therapies. We conducted a retrospective analysis to test the safety and efficacy of a new combination immune therapy in 131 unselected stage IV solid cancer patients with 23 different histological types of cancer who exhausted all conventional treatments. Treatment consisted of locoregional- and whole-body hyperthermia, individually dose adapted interleukin 2 (IL-2) combined with low-dose ipilimumab (0.3 mg/kg) plus nivolumab (0.5 mg/kg). The objective response rate (ORR) was 31.3%, progression-free survival (PFS) was 10 months, survival probabilities at 6 months was 86.7% (95% CI, 81.0-92.8%), at 9 months was 73.5% (95% CI, 66.2-81.7%), at 12 months was 66.5% (95% CI, 58.6-75.4%), while at 24 months survival was 36.6% (95% CI:28.2%; 47.3%). irAEs of World Health Organization (WHO) Toxicity Scale grade 1, 2, 3, and 4 were observed in 23.66%, 16.03%, 6.11%, and 2.29% of patients, respectively. Our results suggest that the irAEs profile of the combined treatment is safer than that of the established protocols without compromising efficacy.

Hospital variation in rates of concurrent fundoplication during gastrostomy enteral access procedures.

BACKGROUND: This study aimed to determine whether (1) the propensity for concurrent fundoplication during gastrostomy varies among hospitals, and (2) postoperative morbidity differs among institutions performing fundoplication more or less frequently. METHODS: Children who underwent gastrostomy with or without concurrent fundoplication were identified in the American College of Surgeons National Surgical Quality Improvement Program Pediatric (ACS-NSQIP-P). A hierarchical multivariate regression modeled the excess effects that hospitals exerted over propensity for concurrent fundoplication adjusting for preoperative clinical variables. Hospitals were designated as low outliers (significantly lower-adjusted odds of concurrent fundoplication than the average hospital with similar patient mix), average hospitals, and high outliers based on their risk-adjusted concurrent fundoplication practice. The postoperative morbidity rates were compared among low-outlier, average, and high-outlier hospitals. RESULTS: Between 2011 and 2013, 3775 children underwent gastrostomy at one of 54 ACS-NSQIP-P participating hospitals. The mean hospital concurrent fundoplication rate was 11.7% (range 0-64%). There was no significant difference in unadjusted morbidity rate in children with concurrent fundoplication, 11.0% compared to 9.7% in children without concurrent fundoplication. After controlling for clinical variables, 8 hospitals were identified as low outliers (fundoplication rate of 0.4%) and 16 hospitals were identified as high outliers (fundoplication rate of 34.6%). The average unadjusted morbidity rate among hospitals with low, average, and high odds of concurrent fundoplication were 9.6, 10.6, and 8.4%, respectively. CONCLUSION: Hospitals vary significantly in propensity for concurrent fundoplication during gastrostomy yet postoperative morbidity does not differ significantly among institutions performing fundoplication more or less frequently.

Patient blood management and the importance of the Transfusion Practitioner role to embed this into practice.

Patient blood management (PBM) is a widely established international initiative, with a multidisciplinary approach to reduce transfusion. The Transfusion Practitioner1 (TP) role is well embedded in the United Kingdom (UK) and Australia. The value of the TP in changing both culture and practice to implement an all-inclusive PBM approach to care will be discussed. The TP role was born from both a safety and haemovigilance culture, where the greatest identified risk to the patient undergoing a transfusion was human error. From this initial trigger for improved safety, the TP role has evolved to a multifaceted, highly specialised role, involved in both PBM and transfusion processes. As the transfusion paradigm shifted from product to patient, the TP role evolved to include PBM, with an emphasis on the patients and the impact transfusion has on them. A multidisciplinary team is required to drive both PBM and transfusion; the TP is recognised as a critical link in the multidisciplinary team. They are seen as a driving force for change, bridging the gap between the laboratory and clinical arenas. The TP plays a vital role in helping establish and embed PBM that improves patient and safety outcomes.

Acylcarnitine Profiles Reflect Metabolic Vulnerability for Necrotizing Enterocolitis in Newborns Born Premature.

OBJECTIVE: To evaluate the association between newborn acylcarnitine profiles and the subsequent development of necrotizing enterocolitis (NEC) with the use of routinely collected newborn screening data in infants born preterm. STUDY DESIGN: A retrospective cohort study was conducted with the use of discharge records for infants born preterm admitted to neonatal intensive care units in California from 2005 to 2009 who had linked state newborn screening results. A model-development cohort of 94 110 preterm births from 2005 to 2008 was used to develop a risk-stratification model that was then applied to a validation cohort of 22 992 births from 2009. RESULTS: Fourteen acylcarnitine levels and acylcarnitine ratios were associated with increased risk of developing NEC. Each log unit increase in C5 and free carnitine /(C16 + 18:1) was associated with a 78% and a 76% increased risk for developing NEC, respectively (OR 1.78, 95% CI 1.53-2.02, and OR 1.76, 95% CI 1.51-2.06). Six acylcarnitine levels, along with birth weight and total parenteral nutrition, identified 89.8% of newborns with NEC in the model-development cohort (area under the curve 0.898, 95% CI 0.889-0.907) and 90.8% of the newborns with NEC in the validation cohort (area under the curve 0.908, 95% CI 0.901-0.930). CONCLUSIONS: Abnormal fatty acid metabolism was associated with prematurity and the development of NEC. Metabolic profiling through newborn screening may serve as an objective biologic surrogate of risk for the development of disease and thus facilitate disease-prevention strategies.

Retrospective forecasting of the 2010-2014 Melbourne influenza seasons using multiple surveillance systems.

Accurate forecasting of seasonal influenza epidemics is of great concern to healthcare providers in temperate climates, since these epidemics vary substantially in their size, timing and duration from year to year, making it a challenge to deliver timely and proportionate responses. Previous studies have shown that Bayesian estimation techniques can accurately predict when an influenza epidemic will peak many weeks in advance, and we have previously tailored these methods for metropolitan Melbourne (Australia) and Google Flu Trends data. Here we extend these methods to clinical observation and laboratory-confirmation data for Melbourne, on the grounds that these data sources provide more accurate characterizations of influenza activity. We show that from each of these data sources we can accurately predict the timing of the epidemic peak 4-6 weeks in advance. We also show that making simultaneous use of multiple surveillance systems to improve forecast skill remains a fundamental challenge. Disparate systems provide complementary characterizations of disease activity, which may or may not be comparable, and it is unclear how a 'ground truth' for evaluating forecasts against these multiple characterizations might be defined. These findings are a significant step towards making optimal use of routine surveillance data for outbreak forecasting.

Altered hippocampal function in major depression despite intact structure and resting perfusion.

BACKGROUND: Hippocampal volume reductions in major depression have been frequently reported. However, evidence for functional abnormalities in the same region in depression has been less clear. We investigated hippocampal function in depression using functional magnetic resonance imaging (fMRI) and neuropsychological tasks tapping spatial memory function, with complementing measures of hippocampal volume and resting blood flow to aid interpretation. METHOD: A total of 20 patients with major depressive disorder (MDD) and a matched group of 20 healthy individuals participated. Participants underwent multimodal magnetic resonance imaging (MRI): fMRI during a spatial memory task, and structural MRI and resting blood flow measurements of the hippocampal region using arterial spin labelling. An offline battery of neuropsychological tests, including several measures of spatial memory, was also completed. RESULTS: The fMRI analysis showed significant group differences in bilateral anterior regions of the hippocampus. While control participants showed task-dependent differences in blood oxygen level-dependent (BOLD) signal, depressed patients did not. No group differences were detected with regard to hippocampal volume or resting blood flow. Patients showed reduced performance in several offline neuropsychological measures. All group differences were independent of differences in hippocampal volume and hippocampal blood flow. CONCLUSIONS: Functional abnormalities of the hippocampus can be observed in patients with MDD even when the volume and resting perfusion in the same region appear normal. This suggests that changes in hippocampal function can be observed independently of structural abnormalities of the hippocampus in depression.

Variation in intraoperative and postoperative red blood cell transfusion in pediatric surgery.

BACKGROUND: Intraoperative and postoperative red blood cell (RBC) transfusions are relatively frequent events tracked in the American College of Surgeons' National Surgical Quality Improvement Program-Pediatric (ACS-NSQIP-P). This study sought to quantify variation in RBC transfusion practices among hospitals. STUDY DESIGN AND METHODS: This is an observational study of children older than 28 days who underwent a general, neurologic, urologic, otolaryngologic, plastic, or orthopedic operation at 50 hospitals in participating in the ACS-NSQIP-P during 2011 to 2012. The primary outcome was whether or not a RBC transfusion was administered from incision time to 72 hours postoperatively. Transfusions of fresh-frozen plasma, cryoprecipitate, and platelets were excluded from data abstraction due the rarity of their administration. A multivariate hierarchical risk-adjustment model estimated the risk-adjusted hospital RBC transfusion odds ratio (OR) and designated hospitals by transfusion practice. RESULTS: The mean RBC transfusion rate was 1.5%. Five preoperative variables were associated with greater than threefold increased odds of having an intraoperative or postoperative RBC transfusion; young age; 29 days to 1 year (OR, 5.9; p < 0.001) and 1 to 2 years (OR, 3.4; p < 0.001); American Society of Anesthesiologists Class IV (OR, 3.2; p < 0.001); procedure linear risk (OR, 3.1; p < 0.001); preoperative septic shock (OR, 14.5; p < 0.001); and preoperative cardiopulmonary resuscitation (OR, 8.1; p < 0.001). Twenty-five hospitals had RBC transfusion practices significantly different than risk-adjusted mean (17 higher and eight lower). CONCLUSION: Intraoperative and postoperative RBC transfusion practices vary widely among hospitals after controlling for patient and procedural characteristics.

Spotlight on platelets: summary of BBTS combined special interest group autumn meeting, November 2015.

This conference first addressed aspects of component quality, highlighting the role of pathogen inactivation, the role of PAS or plasma in prolonging platelet viability and acceptable storage deviations. A series of talks on the medical use of platelets covered the role of platelet transfusion in preventing intracranial haemorrhage, platelet prophylaxis in haematological patients and the new trial of the HLA Matchmaker programme to provide epitope-matched platelets. The session on the surgical use of platelets considered the role of platelet transfusions in patients on anti-platelet agents, major trauma and interventional procedures and also the scope for tests of platelet function to direct therapy. The conference concluded with a panel discussion highlighting key areas of general interest, including the clinical use of platelets and near patient platelet function tests.

Real-time supervised detection of pink areas in dermoscopic images of melanoma: importance of color shades, texture and location.

BACKGROUND/PURPOSE: Early detection of malignant melanoma is an important public health challenge. In the USA, dermatologists are seeing more melanomas at an early stage, before classic melanoma features have become apparent. Pink color is a feature of these early melanomas. If rapid and accurate automatic detection of pink color in these melanomas could be accomplished, there could be significant public health benefits. METHODS: Detection of three shades of pink (light pink, dark pink, and orange pink) was accomplished using color analysis techniques in five color planes (red, green, blue, hue, and saturation). Color shade analysis was performed using a logistic regression model trained with an image set of 60 dermoscopic images of melanoma that contained pink areas. Detected pink shade areas were further analyzed with regard to the location within the lesion, average color parameters over the detected areas, and histogram texture features. RESULTS: Logistic regression analysis of a separate set of 128 melanomas and 128 benign images resulted in up to 87.9% accuracy in discriminating melanoma from benign lesions measured using area under the receiver operating characteristic curve. The accuracy in this model decreased when parameters for individual shades, texture, or shade location within the lesion were omitted. CONCLUSION: Texture, color, and lesion location analysis applied to multiple shades of pink can assist in melanoma detection. When any of these three details: color location, shade analysis, or texture analysis were omitted from the model, accuracy in separating melanoma from benign lesions was lowered. Separation of colors into shades and further details that enhance the characterization of these color shades are needed for optimal discrimination of melanoma from benign lesions.

Urine biomarkers for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in premature neonates. Despite decades of investigation, treating clinicians are still not able to determine which premature infants are at greatest risk of developing NEC and which of the affected infants will develop severe NEC requiring operation. A biomarker is a specific molecular indicator that can be used to identify or measure the progress of a disease. Many potential biomarkers have been studied for their potential relevance to NEC. Those showing promise include C-reactive protein, intestinal fatty acid-binding protein, platelet-activating factor and many others. None to date have achieved sufficient predictive value to be clinically useful. Distinguishing between the specific changes in NEC and the non-specific inflammation of sepsis has proven challenging. Urine is a particularly attractive site for potential biomarkers. It can be collected readily and non-invasively, and it is a rich source of both proteins and peptides. Preliminary work has revealed some promising biomarkers of NEC in urine. Combined with clinical data, they have been shown to be highly predictive in small series of patients. Advances in high-throughput molecular analysis have opened the door to finding biomarkers that may meaningfully improve the outcome of infants at risk for NEC.

A laser driven pulsed X-ray backscatter technique for enhanced penetrative imaging.

X-ray backscatter imaging can be used for a wide range of imaging applications, in particular for industrial inspection and portal security. Currently, the application of this imaging technique to the detection of landmines is limited due to the surrounding sand or soil strongly attenuating the 10s to 100s of keV X-rays required for backscatter imaging. Here, we introduce a new approach involving a 140 MeV short-pulse (< 100 fs) electron beam generated by laser wakefield acceleration to probe the sample, which produces Bremsstrahlung X-rays within the sample enabling greater depths to be imaged. A variety of detector and scintillator configurations are examined, with the best time response seen from an absorptive coated BaF2 scintillator with a bandpass filter to remove the slow scintillation emission components. An X-ray backscatter image of an array of different density and atomic number items is demonstrated. The use of a compact laser wakefield accelerator to generate the electron source, combined with the rapid development of more compact, efficient and higher repetition rate high power laser systems will make this system feasible for applications in the field. Content includes material subject to Dstl (c) Crown copyright (2014). Licensed under the terms of the Open Government Licence except where otherwise stated. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: psi@ nationalarchives.gsi.gov.uk.

A novel urine peptide biomarker-based algorithm for the prognosis of necrotising enterocolitis in human infants.

OBJECTIVE: Necrotising enterocolitis (NEC) is a major source of neonatal morbidity and mortality. The management of infants with NEC is currently complicated by our inability to accurately identify those at risk for progression of disease prior to the development of irreversible intestinal necrosis. We hypothesised that integrated analysis of clinical parameters in combination with urine peptide biomarkers would lead to improved prognostic accuracy in the NEC population. DESIGN: Infants under suspicion of having NEC (n=550) were prospectively enrolled from a consortium consisting of eight university-based paediatric teaching hospitals. Twenty-seven clinical parameters were used to construct a multivariate predictor of NEC progression. Liquid chromatography/mass spectrometry was used to profile the urine peptidomes from a subset of this population (n=65) to discover novel biomarkers of NEC progression. An ensemble model for the prediction of disease progression was then created using clinical and biomarker data. RESULTS: The use of clinical parameters alone resulted in a receiver-operator characteristic curve with an area under the curve of 0.817 and left 40.1% of all patients in an 'indeterminate' risk group. Three validated urine peptide biomarkers (fibrinogen peptides: FGA1826, FGA1883 and FGA2659) produced a receiver-operator characteristic area under the curve of 0.856. The integration of clinical parameters with urine biomarkers in an ensemble model resulted in the correct prediction of NEC outcomes in all cases tested. CONCLUSIONS: Ensemble modelling combining clinical parameters with biomarker analysis dramatically improves our ability to identify the population at risk for developing progressive NEC.

Urine protein biomarkers for the diagnosis and prognosis of necrotizing enterocolitis in infants.

OBJECTIVES: To test the hypothesis that an exploratory proteomics analysis of urine proteins with subsequent development of validated urine biomarker panels would produce molecular classifiers for both the diagnosis and prognosis of infants with necrotizing enterocolitis (NEC). STUDY DESIGN: Urine samples were collected from 119 premature infants (85 NEC, 17 sepsis, 17 control) at the time of initial clinical concern for disease. The urine from 59 infants was used for candidate biomarker discovery by liquid chromatography/mass spectrometry. The remaining 60 samples were subject to enzyme-linked immunosorbent assay for quantitative biomarker validation. RESULTS: A panel of 7 biomarkers (alpha-2-macroglobulin-like protein 1, cluster of differentiation protein 14, cystatin 3, fibrinogen alpha chain, pigment epithelium-derived factor, retinol binding protein 4, and vasolin) was identified by liquid chromatography/mass spectrometry and subsequently validated by enzyme-linked immunosorbent assay. These proteins were consistently found to be either up- or down-regulated depending on the presence, absence, or severity of disease. Biomarker panel validation resulted in a receiver-operator characteristic area under the curve of 98.2% for NEC vs sepsis and an area under the curve of 98.4% for medical NEC vs surgical NEC. CONCLUSIONS: We identified 7 urine proteins capable of providing highly accurate diagnostic and prognostic information for infants with suspected NEC. This work represents a novel approach to improving the efficiency with which we diagnose early NEC and identify those at risk for developing severe, or surgical, disease.

DAS181 treatment of hematopoietic stem cell transplant patients with parainfluenza virus lung disease requiring mechanical ventilation.

Parainfluenza infection is a cause of significant morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) patients. DAS181 is a novel antiviral agent with activity against influenza and parainfluenza. We report the first 2 cases, to our knowledge, of successful DAS181 use in ventilated HSCT patients with severe parainfluenza lung disease.

Current concepts in the surgical approach to necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most common surgical emergency occurring in neonatal intensive care unit (NICU) patients. Among patients with NEC, those that require surgery experience the poorest outcomes and highest mortality. Surgical intervention, while attempting to address the intestinal injury and ongoing mulitfactorial physiologic insults in NEC is associated with its own stresses that may compound the ongoing physiologic derangement. Surgery is thus reserved for those patients with clear indication for intervention such as pneumoperitoneum, confirmed stool or pus in the peritoneal cavity, or worsening clinical status. The purpose of this review is to briefly describe the physiologic stress induced by surgical intervention in the preterm, low birth weight patient with NEC and to provide a contemporary overview of available surgical management options for NEC. The optimal surgical plan employed is strongly influenced by clinical judgment and theoretical benefits in terms of minimizing physiologic stressors while providing temporary and/or definitive treatment in a timely fashion. While the choice of operation has not been shown to have a significant effect on any clinically important outcomes, ongoing investigations continue to study both short and long-term outcomes in patients with NEC.

Allergic bronchopulmonary aspergillosis: review of literature and proposal of new diagnostic and classification criteria.

Allergic bronchopulmonary aspergillosis (ABPA) is an immunological pulmonary disorder caused by hypersensitivity to Aspergillus fumigatus, manifesting with poorly controlled asthma, recurrent pulmonary infiltrates and bronchiectasis. There are estimated to be in excess of four million patients affected world-wide. The importance of recognizing ABPA relates to the improvement of patient symptoms, and delay in development or prevention of bronchiectasis, one manifestation of permanent lung damage in ABPA. Environmental factors may not be the only pathogenetic factors because not all asthmatics develop ABPA despite being exposed to the same environment. Allergic bronchopulmonary aspergillosis is probably a polygenic disorder, which does not remit completely once expressed, although long-term remissions do occur. In a genetically predisposed individual, inhaled conidia of A. fumigatus germinate into hyphae with release of antigens that activate the innate and adaptive immune responses (Th2 CD4(+) T cell responses) of the lung. The International Society for Human and Animal Mycology (ISHAM) has constituted a working group on ABPA complicating asthma (www.abpaworkinggroup.org), which convened an international conference to summarize the current state of knowledge, and formulate consensus-based guidelines for diagnosis and therapy. New diagnosis and staging criteria for ABPA are proposed. Although a small number of randomized controlled trials have been conducted, long-term management remains poorly studied. Primary therapy consists of oral corticosteroids to control exacerbations, itraconazole as a steroid-sparing agent and optimized asthma therapy. Uncertainties surround the prevention and management of bronchiectasis, chronic pulmonary aspergillosis and aspergilloma as complications, concurrent rhinosinusitis and environmental control. There is need for new oral antifungal agents and immunomodulatory therapy.

Neonatal mouse-derived engineered cardiac tissue: a novel model system for studying genetic heart disease.

RATIONALE: Cardiomyocytes cultured in a mechanically active 3-dimensional configuration can be used for studies that correlate contractile performance to cellular physiology. Current engineered cardiac tissue (ECT) models use cells derived from either rat or chick hearts. Development of a murine ECT would provide access to many existing models of cardiac disease and open the possibility of performing targeted genetic manipulation with the ability to directly assess contractile and molecular variables. OBJECTIVE: To generate, characterize, and validate mouse ECT with a physiologically relevant model of hypertrophic cardiomyopathy. METHODS AND RESULTS: We generated mechanically integrated ECT using isolated neonatal mouse cardiac cells derived from both wild-type and myosin-binding protein C (cMyBP-C)-null mouse hearts. The murine ECTs produced consistent contractile forces that followed the Frank-Starling law and accepted physiological pacing. cMyBP-C-null ECTs showed characteristic acceleration of contraction kinetics. Adenovirus-mediated expression of human cMyBP-C in murine cMyBP-C-null ECT restored contractile properties to levels indistinguishable from those of wild-type ECT. Importantly, the cardiomyocytes used to construct the cMyBP-C(-/-) ECT had yet to undergo the significant hypertrophic remodeling that occurs in vivo. Thus, this murine ECT model reveals a contractile phenotype that is specific to the genetic mutation rather than to secondary remodeling events. CONCLUSIONS: Data presented here show mouse ECT to be an efficient and cost-effective platform to study the primary effects of genetic manipulation on cardiac contractile function. This model provides a previously unavailable tool to study specific sarcomeric protein mutations in an intact mammalian muscle system.

DAS181 treatment of severe parainfluenza type 3 pneumonia in a lung transplant recipient.

Parainfluenza virus (PIV) may cause life-threatening pneumonia in lung transplant patients and there are no proven effective therapies. We report the use of inhaled DAS181, a novel sialidase fusion protein, to treat severe PIV type 3 pneumonia in a lung transplant patient. Treatment was well tolerated and associated with improvement in oxygenation and symptoms, along with rapid clearance of PIV. DAS181 should be systematically evaluated for treatment of PIV infection in transplant recipients.