Direct detection of dark matter-APPEC committee report.

This report provides an extensive review of the experimental programme of direct detection searches of particle dark matter. It focuses mostly on European efforts, both current and planned, but does it within a broader context of a worldwide activity in the field. It aims at identifying the virtues, opportunities and challenges associated with the different experimental approaches and search techniques. It presents scientific and technological synergies, both existing and emerging, with some other areas of particle physics, notably collider and neutrino programmes, and beyond. It addresses the issue of infrastructure in light of the growing needs and challenges of the different experimental searches. Finally, the report makes a number of recommendations from the perspective of a long-term future of the field. They are introduced, along with some justification, in the opening overview and recommendations section and are next summarised at the end of the report. Overall, we recommend that the direct search for dark matter particle interactions with a detector target should be given top priority in astroparticle physics, and in all particle physics, and beyond, as a positive measurement will provide the most unambiguous confirmation of the particle nature of dark matter in the Universe.

The First Treatment for PKU: The Pioneers-Birmingham 1951.

Prior to the introduction of newborn screening, Phenylketonuria (PKU) was a devastating disorder with affected individuals usually committed to a life in care in large institutions (asylums). Newborn screening only began after it was shown that those with PKU could be treated with a modified diet and could subsequently lead normal lives. The first production of a diet and the demonstration of its effectiveness was thus a key milestone in the history of both PKU and newborn screening, and took place in Birmingham, UK, in 1951. The pioneers were a two-year-old girl called Sheila Jones, her mother Mary, and three dedicated professionals at Birmingham Children's Hospital: Evelyn Hickmans, John Gerrard and Horst Bickel. Together, they changed the course of PKU for those across the world. This review summarises the history and achievements of this team who opened the door to PKU treatment and the introduction of newborn screening.

Atypical ductal hyperplasia on core needle biopsy: Surgical outcomes of 200 consecutive cases from a high-volume breast program.

Atypical ductal hyperplasia (ADH) is an indication for excisional biopsy to rule out occult breast cancer. We analyzed pathological findings on excisional biopsy for ADH diagnosed in a high volume breast center equipped with digital tomosynthesis. Two hundred consecutive patients were diagnosed with ADH on core biopsy with radiographic concordance followed by excisional biopsy. On excisional biopsy, 33 patients (16.5%) were diagnosed with DCIS or invasive breast cancer. Patients with a concurrent diagnosis of papilloma had a higher risk of upstaging on both univariate and multivariate analysis (41.7% vs. 14.9%, p=0.015). No other statistically significant predictors of upgrading were identified (p>0.05).

Atypical lobular hyperplasia on core needle biopsy: contemporary results from a large community hospital breast program.

PURPOSE: The management of biopsy proven atypical lobular hyperplasia (ALH) is controversial. Although upgrade rates are low, excisional biopsy is often performed to rule out occult breast cancer. METHODS: In this study, we analyzed our experience with excisional biopsy for ALH diagnosed in the digital tomosynthesis era with radiographic concordance in the community hospital setting. This study included 93 consecutive patients diagnosed with pure ALH on core biopsy from January 2013-December 2017 who underwent subsequent excisional biopsy. Potential clinical, radiographic and pathologic predictors of upgrading were analyzed. RESULTS: At the time of excisional biopsy, five patients (5.4%) were upgraded to DCIS or invasive breast cancer. There was also a trend towards higher upgrade rates in patients with contralateral breast cancer (p = 0.06), biopsy performed by ultrasound or MRI (p = 0.07) and extensive ALH (p = 0.10). Other clinical, radiographic and pathologic variables were not predictive of upgrade rate (p > 0.1 for all). CONCLUSION: Patients with pure ALH with radiographic concordance have a low risk of pathologic upgrading on excisional biopsy. Potential predictors of upgrade rate warrant further analysis in a larger dataset.

Model independent determination of the dark matter mass from direct detection experiments.

Determining the dark matter (DM) mass is of paramount importance for understanding dark matter. We present a novel parametrization of the DM speed distribution which will allow the DM mass to be accurately measured using data from weakly interacting massive particle (WIMP) direct detection experiments. Specifically, we parametrize the natural logarithm of the speed distribution as a polynomial in the speed v. We demonstrate, using mock data from upcoming experiments, that by fitting the WIMP mass and interaction cross section, along with the polynomial coefficients, we can accurately reconstruct both the WIMP mass and speed distribution. This new method is the first demonstration that an accurate, unbiased reconstruction of the WIMP mass is possible without prior assumptions about the distribution function. We anticipate that this technique will be invaluable in the analysis of future experimental data.

Newborn screening for medium chain acyl-CoA dehydrogenase deficiency in England: prevalence, predictive value and test validity based on 1.5 million screened babies.

BACKGROUND: Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is a rare, life-threatening condition. Early diagnosis by screening asymptomatic newborns may improve outcome, but the benefit to newborns identified with variants not encountered clinically is uncertain. OBJECTIVE: To estimate, overall and by ethnic group: screen-positive prevalence and predictive value (PPV); MCADD prevalence; proportion MCADD variants detected of predicted definite or uncertain clinical importance. SETTING: All births in areas of high ethnic minority prevalence in England. METHODS: Prospective multicentre pilot screening service; testing at age five to eight days; standardized screening, diagnostic and management protocols; independent expert review of screen-positive cases to assign MCADD diagnosis and predicted clinical importance (definite or uncertain). RESULTS: Approximately 1.5 million babies (79% white; 10% Asian) were screened. MCADD was confirmed in 147 of 190 babies with a positive screening result (screen-positive prevalence: 1.20 per 10,000; MCADD prevalence: 0.94 per 10,000; PPV 77% [95% CI 71-83]), comprising 103 (70%) with MCADD variants of definite clinical importance (95 white [95%]; 2 Asian [2%]) and 44 (30%) with variants of uncertain clinical importance (29 white [67%]; 12 Asian [28%]). CONCLUSION: One baby in every 10,000 born in England is diagnosed with MCADD by newborn screening; around 60 babies each year. While the majority of MCADD variants detected are predicted to be of definite clinical importance, this varies according to ethnic group, with variants of uncertain importance most commonly found in Asian babies. These findings provide support for MCADD screening but highlight the need to take account of the ethnic diversity of the population tested at implementation.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer.

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.

17Beta-estradiol elevates cGMP and, via plasma membrane recruitment of protein kinase GIalpha, stimulates Ca2+ efflux from rat hepatocytes.

Rapid non-genomic effects of 17beta-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17beta-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17beta-estradiol, but not 17alpha-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Ialpha, stimulates plasma membrane Ca(2+)-ATPase-mediated Ca(2+) efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17beta-estradiol-BSA conjugate, suggesting that 17beta-estradiol acts at the extracellular face of the plasma membrane. We also show that 17beta-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative gamma-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17beta-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the gamma-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.

Relationship of octanoylcarnitine concentrations to age at sampling in unaffected newborns screened for medium-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Although octanoylcarnitine (C8) concentrations measured from newborn screening dried blood spots are used to identify those at high risk of medium-chain acyl-CoA dehydrogenase deficiency (MCADD), age-related reference values are currently not available for unaffected newborn populations. Because age at sampling may vary within and between screening programs, variations in C8 concentrations by age may affect screening program performance. We determined whether C8 concentrations vary by age at sampling, sex, birth weight, or gestational age in unaffected newborns. METHODS: We analyzed C8 concentrations from 227,098 unaffected newborns, including 179,729 from 6 English laboratories participating in a multicenter study and 47,369 from the single laboratory serving the New South Wales (NSW) Newborn Screening Program in Australia. In England, the majority of samples were collected at age 5-8 days and analyzed underivatized by use of tandem mass spectrometry (MS/MS); in NSW, samples were obtained at a median age of 3 days and analyzed derivatized by MS/MS. Information on infants' sex, birth weight, gestation, hospitalization, and transfusion status was recorded at time of sampling. RESULTS: C8 concentrations did not vary significantly by age at sampling, sex, birth weight, or gestational age and remained relatively constant during the first 2 weeks of life in unaffected babies being screened for MCADD. CONCLUSIONS: Newborn MCADD screening programs using this biomarker for screening samples collected after the first day and during the first 14 days of life do not need to adjust cutoff values to account for postnatal age, prematurity, or size at birth.

Limitations of the murine nose in the development of nonviral airway gene transfer.

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.

Identification and functional characterization of cytoplasmic determinants of plasmid DNA nuclear import.

Import of exogenous plasmid DNA (pDNA) into mammalian cell nuclei represents a key intracellular obstacle to efficient non-viral gene delivery. This includes access of the pDNA to the nuclei of non-dividing cells where the presence of an intact nuclear membrane is limiting for gene transfer. Here we identify, isolate, and characterize, cytoplasmic determinants of pDNA nuclear import into digitonin-permeabilized HeLa cells. Depletion of putative DNA-binding proteins, on the basis of their ability to bind immobilized pDNA, abolished pDNA nuclear import supporting the critical role of cytoplasmic factors in this process. Elution of pDNA-bound proteins, followed by two-dimensional sodium dodecyl polyacrylamide gel electrophoresis identified several candidate DNA shuttle proteins. We show that two of these, NM23-H2, a ubiquitous c-Myc transcription-activating nucleoside diphosphate kinase, and the core histone H2B can both reconstitute pDNA nuclear import. Further, we demonstrate a significant increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from two of the DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10. These data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus using the minimal import machinery. Importantly, increasing the binding of pDNA to shuttle proteins by re-engineering reporter plasmids with shuttle binding sequences enhances gene transfer. Increasing the potential for exogenously added pDNA to bind intracellular transport cofactors may enhance the potency of non-viral gene transfer.

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression.

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (>or=56 d) in vivo transgene expression in the absence of lung inflammation.

Information transfer in signaling pathways: a study using coupled simulated and experimental data.

BACKGROUND: The topology of signaling cascades has been studied in quite some detail. However, how information is processed exactly is still relatively unknown. Since quite diverse information has to be transported by one and the same signaling cascade (e.g. in case of different agonists), it is clear that the underlying mechanism is more complex than a simple binary switch which relies on the mere presence or absence of a particular species. Therefore, finding means to analyze the information transferred will help in deciphering how information is processed exactly in the cell. Using the information-theoretic measure transfer entropy, we studied the properties of information transfer in an example case, namely calcium signaling under different cellular conditions. Transfer entropy is an asymmetric and dynamic measure of the dependence of two (nonlinear) stochastic processes. We used calcium signaling since it is a well-studied example of complex cellular signaling. It has been suggested that specific information is encoded in the amplitude, frequency and waveform of the oscillatory Ca(2+)-signal. RESULTS: We set up a computational framework to study information transfer, e.g. for calcium signaling at different levels of activation and different particle numbers in the system. We stochastically coupled simulated and experimentally measured calcium signals to simulated target proteins and used kernel density methods to estimate the transfer entropy from these bivariate time series. We found that, most of the time, the transfer entropy increases with increasing particle numbers. In systems with only few particles, faithful information transfer is hampered by random fluctuations. The transfer entropy also seems to be slightly correlated to the complexity (spiking, bursting or irregular oscillations) of the signal. Finally, we discuss a number of peculiarities of our approach in detail. CONCLUSION: This study presents the first application of transfer entropy to biochemical signaling pathways. We could quantify the information transferred from simulated/experimentally measured calcium signals to a target enzyme under different cellular conditions. Our approach, comprising stochastic coupling and using the information-theoretic measure transfer entropy, could also be a valuable tool for the analysis of other signaling pathways.

ANP stimulates hepatocyte Ca2+ efflux via plasma membrane recruitment of PKGIalpha.

In rat hepatocytes, atrial natriuretic peptide (ANP) elevates cGMP through activation of particulate guanylyl cyclase and attenuates Ca(2+) signals by stimulating net plasma membrane Ca(2+) efflux. We show here that ANP-stimulated hepatocyte Ca(2+) efflux is mediated by protein kinase G (PKG) isotype I. Furthermore, we show that ANP recruits endogenous PKGIalpha, but not PKGIbeta, to the plasma membrane. These effects are mimicked by 8-bromo-cGMP, but not by the soluble guanylyl cyclase activators, sodium nitroprusside and YC-1. We propose that ANP, through localized cGMP elevation, promotes plasma membrane recruitment of PKGIalpha, which, in turn, stimulates Ca(2+) efflux.

Establishing the stochastic nature of intracellular calcium oscillations from experimental data.

Calcium has been established as a key messenger in both intra- and intercellular signaling. Experimentally observed intracellular calcium responses to different agonists show a variety of behaviors from simple spiking to complex oscillatory regimes. Here we study typical experimental traces of calcium oscillations in hepatocytes obtained in response to phenylephrine and ATP. The traces were analyzed with methods of nonlinear time series analysis in order to determine the stochastic/deterministic nature of the intracellular calcium oscillations. Despite the fact that the oscillations appear, visually, to be deterministic yet perturbed by noise, our analyses provide strong evidence that the measured calcium traces in hepatocytes are prevalently of stochastic nature. In particular, bursting calcium oscillations are temporally correlated Gaussian series distorted by a monotonic, instantaneous, time-independent function, whilst the spiking behavior appears to have a dynamical nonlinear component whereby the overall determinism level is still low. The biological importance of this finding is discussed in relation to the mechanisms incorporated in mathematical models as well as the role of stochasticity and determinism at cellular and tissue levels which resemble typical statistical and thermodynamic effects in physics.

Spatio-temporal modelling explains the effect of reduced plasma membrane Ca2+ efflux on intracellular Ca2+ oscillations in hepatocytes.

In many non-excitable eukaryotic cells, including hepatocytes, Ca(2+) oscillations play a key role in intra- and intercellular signalling, thus regulating many cellular processes from fertilisation to death. Therefore, understanding the mechanisms underlying these oscillations, and consequently understanding how they may be regulated, is of great interest. In this paper, we study the influence of reduced Ca(2+) plasma membrane efflux on Ca(2+) oscillations in hepatocytes. Our previous experiments with carboxyeosin show that a reduced plasma membrane Ca(2+) efflux increases the frequency of Ca(2+) oscillations, but does not affect the duration of individual transients. This phenomenon can be best explained by taking into account not only the temporal, but also the spatial dynamics underlying the generation of Ca(2+) oscillations in the cell. Here we divide the cell into a grid of elements and treat the Ca(2+) dynamics as a spatio-temporal phenomenon. By converting an existing temporal model into a spatio-temporal one, we obtain theoretical predictions that are in much better agreement with the experimental observations.

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux.

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.

Quantitation of urinary glycosaminoglycans using dimethylene blue as a screening technique for the diagnosis of mucopolysaccharidoses: an evaluation.

BACKGROUND: The mucopolysaccharidoses (MPSs) are a group of inherited disorders due to defects in lysosomal enzymes catalysing the breakdown of glycosaminoglycans (GAGs). Usually they are detected using techniques to separate the accumulating GAGs in urine. However more recently an improved dye-based method measuring total urine GAG concentrations has become available. We have evaluated this method in the context of its application as a general screening technique in a routine metabolic laboratory. METHOD: An automated method for the quantitation of urinary GAGs using dimethylene blue was developed on a Cobas Fara analyser and evaluated in terms of its specificity, sensitivity and value in the diagnosis of the MPSs. The test was applied to 6156 anonymized urine samples received for tests for inherited metabolic diseases and to 121 samples from 85 patients with a variety of proven MPSs. RESULTS: A substantial number of samples from unaffected individuals gave abnormal results while a significant number of samples from patients with MPSs gave results within normal limits. CONCLUSION: In the context of our laboratory, it was not appropriate for use as a screening technique when applied to all specimens received for metabolic tests or as a first-line screening test for samples where mucopolysaccharide analysis was requested. However it was retained and used in parallel with cellulose acetate electrophoresis to aid interpretation.

The sweat test for the diagnosis of cystic fibrosis--a personal experience of guideline production.

The need for evidence-based guidelines as to how to conduct the sweat test in the UK was highlighted in 2000 by the National External Quality Assessment Scheme Specialist Advisory Committee. A Guideline Group supported by several professional bodies was subsequently convened and formal evidence-based guidelines were produced and published in 2003. The process of undertaking guideline production is summarized here, together with the author's personal opinions about the experience and highlights of the shortcomings and lessons learnt from the project. In summary, production of evidence-based guidelines is a long process and is only worth the effort (both man hours and financial) if the recommendations are subsequently adopted into clinical practice and in turn improve patient care. The emphasis for the future should be to assess the impact and value of evidence-based guidelines and promote their introduction into local practice as part of care pathways. A growing number of guideline initiatives emphasizes the importance of and a need for an overarching coordinating structure to overcome problems of duplication and enable interested professionals from different disciplines to work on related initiatives.

Guidelines for the performance of the sweat test for the diagnosis of cystic fibrosis.

A multidisciplinary group (representing various professional bodies and supported by the Cystic Fibrosis Trust) has developed evidence-based guidelines for the performance of the sweat test in the UK. The guidelines cover patient information, subject suitability, sweat collection, sweat analysis, quality, interpretation of results, and responsibility for testing and training. The guidelines were produced following a detailed literature search by the process described by the Royal College of Paediatrics and Child Health (RCPCH), using the Scottish Intercollegiate Guidelines Network 1 (SIGN 1) criteria to grade the evidence. Recommendations are graded A, B, or C, depending on the level of evidence. The grade B recommendations (there were no grade A recommendations) were subsequently appraised and endorsed as part of the RCPCH process, according to Appraisal of Guidelines for Research and Evaluation in Europe (AGREE). The recommendations are summarized in tabular form representing the final version incorporating the comments from the appraisal process. Both the appraisal comments and the full evidence base can be found on www.rcpch.ac.uk/publications/clinical_docs.html. The full guidelines can also be found on http://www.ukneqas.org.uk/guidelines.htm.