Background
We sought to determine if next‐generation sequencing (NGS) of microbial cell‐free DNA (cfDNA) in plasma would detect pathogens in pediatric patients at risk for invasive fungal disease ...(IFD).
Procedures
Pediatric hematology, oncology, and stem cell transplant patients deemed at risk for new IFD had blood samples drawn at three time‐points separated by 1‐month intervals. The primary outcome measure was detection of fungal pathogens compared to standard clinical testing. Secondary outcomes included identification of other infectious pathogens, relationship to European Organization for Research and Treatment of Cancer's Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases’ Mycoses Study Group (EORTC/MSG) guidelines, and assessment of antifungal therapy.
Results
NGS identified fungal pathogens in seven of 40 at‐risk patients for IFD and results were identical in four of six proven cases, including Aspergillus fumigatus by lung biopsy, Candida albicans by blood or pancreatic pseudocyst cultures, and Rhizopus delemar by skin biopsy. Rhizopus oryzae identified on skin biopsy and A. fumigatus isolated on day 27 of 28 of culture from lung biopsy were not detected by cfDNA NGS, possibly due to lack of bloodstream penetration and questionable pathogenicity, respectively. Numerous DNA viruses were detected in patients with prolonged febrile neutropenia or abnormal imaging. Extended antifungal therapy was used in 73% of patients. Follow‐up cfDNA sequencing in patients who were positive at enrollment was negative at 1 and 2 months.
Conclusions
cfDNA NGS detected fungal pathogens from blood confirming its potential to guide treatment decisions in pediatric patients at risk for IFD and limit excessive empiric antifungal use. Future studies are needed to better understand the sensitivity and specificity of this approach.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals ...with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The Bactrocera dorsalis complex of tropical fruit flies (Diptera: Tephritidae: Dacinae) contains 75 described species, largely endemic to Southeast Asia. Within the complex are a small number of ...polyphagous pests of international significance, including B. dorsalis sensu stricto, B. papayae, B. carambolae, and B. philippinensis. Most species within the complex were described in 1994 and since then substantial research has been undertaken in developing morphological and molecular diagnostic techniques for their recognition. Such techniques can now resolve most taxa adequately. Genetic evidence suggests that the complex has evolved in only the last few million years, and development of a phylogeny of the group is considered a high priority to provide a framework for future evolutionary and ecological studies. As model systems, mating studies on B. dorsalis s.s. and B. cacuminata have substantially advanced our understanding of insect use of plant-derived chemicals for mating, but such studies have not been applied to help resolve the limits of biological species within the complex. Although they are commonly regarded as major pests, there is little published evidence documenting economic losses caused by flies of the B. dorsalis complex. Quantification of economic losses caused by B. dorsalis complex species is urgently needed to prioritize research for quarantine and management. Although they have been documented as invaders, relatively little work has been done on the invasion biology of the complex and this is an area warranting further work.
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► Bactrocera was paraphyletic; the subgenus group Zeugodacus was instead sister to Dacus and should be raised to genus level. ► Subgeneric taxonomy, particularly within Dacus, ...requires extensive revision. ► Phylogenetic pattern largely follows that predicted by an out-of-India hypothesis. ► However, Dacus diversified earlier than predicted, implying two migrations out of India: Dacus early, Bactrocera late. ► A new dispersal pathway into Africa is proposed for Dacus via Madagascar, with two subsequent migrations back into Asia.
With well over 700 species, the Tribe Dacini is one of the most species-rich clades within the dipteran family Tephritidae, the true fruit flies. Nearly all Dacini belong to one of two very large genera, Dacus Fabricius and Bactrocera Macquart. The distribution of the genera overlap in or around the Indian subcontinent, but the greatest diversity of Dacus is in Africa and the greatest diversity of Bactrocera is in south-east Asia and the Pacific. The monophyly of these two genera has not been rigorously established, with previous phylogenies only including a small number of species and always heavily biased to one genus over the other. Moreover, the subgeneric taxonomy within both genera is complex and the monophyly of many subgenera has not been explicitly tested. Previous hypotheses about the biogeography of the Dacini based on morphological reviews and current distributions of taxa have invoked an out-of-India hypothesis; however this has not been tested in a phylogenetic framework. We attempted to resolve these issues with a dated, molecular phylogeny of 125 Dacini species generated using 16S, COI, COII and white eye genes. The phylogeny shows that Bactrocera is not monophyletic, but rather consists of two major clades: Bactrocera s.s. and the ‘Zeugodacus group of subgenera’ (a recognised, but informal taxonomic grouping of 15 Bactrocera subgenera). This ‘Zeugodacus’ clade is the sister group to Dacus, not Bactrocera and, based on current distributions, split from Dacus before that genus moved into Africa. We recommend that taxonomic consideration be given to raising Zeugodacus to genus level. Supportive of predictions following from the out-of-India hypothesis, the first common ancestor of the Dacini arose in the mid-Cretaceous approximately 80mya. Major divergence events occurred during the Indian rafting period and diversification of Bactrocera apparently did not begin until after India docked with Eurasia (50–35mya). In contrast, diversification in Dacus, at approximately 65mya, apparently began much earlier than predicted by the out-of-India hypothesis, suggesting that, if the Dacini arose on the Indian plate, then ancestral Dacus may have left the plate in the mid to late Cretaceous via the well documented India–Madagascar–Africa migration route. We conclude that the phylogeny does not disprove the predictions of an out-of-India hypothesis for the Dacini, although modification of the original hypothesis is required.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral ...CFTR gene therapy in patients with cystic fibrosis.
We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50–90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene–liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867.
Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups.
Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials.
Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.
Rare earth element (REE) fluorocarbonate mineralization occurs in lacustrine shales in the Lower Devonian Rhynie chert, Aberdeenshire, UK, preserved by hot spring silicification. Mineralization ...follows a combination of first-cycle erosion of granite to yield detrital monazite grains, bioweathering of the monazite to liberate REEs, and interaction with fluorine-rich hot spring fluids in an alkaline sedimentary environment. The mineral composition of most of the fluorocarbonates is referable to synchysite. Mineralization occurs at the surface, and the host shales subsequently experience maximum temperatures of about 110 ℃. Most fluorocarbonate mineralization originates at much higher temperatures, but the Rhynie occurrence emphasizes that low-temperature deposits are possible when both fluorine and REEs are available from granite into the sedimentary environment.
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CEKLJ, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Estimates of the effectiveness of influenza vaccines in older adults may be biased because of difficulties identifying and adjusting for confounders of the vaccine-outcome association. We estimated ...vaccine effectiveness for prevention of serious influenza complications among older persons by using methods to account for underlying differences in risk for these complications.
We conducted a retrospective cohort study among Ontario residents aged ≥ 65 years from September 1993 through September 2008. We linked weekly vaccination, hospitalization, and death records for 1.4 million community-dwelling persons aged ≥ 65 years. Vaccine effectiveness was estimated by comparing ratios of outcome rates during weeks of high versus low influenza activity (defined by viral surveillance data) among vaccinated and unvaccinated subjects by using log-linear regression models that accounted for temperature and time trends with natural spline functions. Effectiveness was estimated for three influenza-associated outcomes: all-cause deaths, deaths occurring within 30 days of pneumonia/influenza hospitalizations, and pneumonia/influenza hospitalizations.
During weeks when 5% of respiratory specimens tested positive for influenza A, vaccine effectiveness among persons aged ≥ 65 years was 22% (95% confidence interval CI, -6%-42%) for all influenza-associated deaths, 25% (95% CI, 13%-37%) for deaths occurring within 30 days after an influenza-associated pneumonia/influenza hospitalization, and 19% (95% CI, 4%-31%) for influenza-associated pneumonia/influenza hospitalizations. Because small proportions of deaths, deaths after pneumonia/influenza hospitalizations, and pneumonia/influenza hospitalizations were associated with influenza virus circulation, we estimated that vaccination prevented 1.6%, 4.8%, and 4.1% of these outcomes, respectively.
By using confounding-reducing techniques with 15 years of provincial-level data including vaccination and health outcomes, we estimated that influenza vaccination prevented ~4% of influenza-associated hospitalizations and deaths occurring after hospitalizations among older adults in Ontario.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In many human diseases, associated genetic changes tend to occur within noncoding regions, whose effect might be related to transcriptional control. A central goal in human genetics is to understand ...the function of such noncoding regions: given a region that is statistically associated with changes in gene expression (expression quantitative trait locus eQTL), does it in fact play a regulatory role? And if so, how is this role “coded” in its sequence? These questions were the subject of the Critical Assessment of Genome Interpretation eQTL challenge. Participants were given a set of sequences that flank eQTLs in humans and were asked to predict whether these are capable of regulating transcription (as evaluated by massively parallel reporter assays), and whether this capability changes between alternative alleles. Here, we report lessons learned from this community effort. By inspecting predictive properties in isolation, and conducting meta‐analysis over the competing methods, we find that using chromatin accessibility and transcription factor binding as features in an ensemble of classifiers or regression models leads to the most accurate results. We then characterize the loci that are harder to predict, putting the spotlight on areas of weakness, which we expect to be the subject of future studies.
Deciphering the functionality of the non‐coding genome has been the focus of many recent studies, aiming to annotate regulatory regions and understand their specific role in disease and other phenotypes. The goal of the CAGI eQTL challenge was to predict the activity of candidate genomic regions (experimentally evaluated by massively parallel reporter assays). Our meta‐analysis of competing submissions highlights features and models that lead to accurate prediction and points to areas for improvement.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Gene therapy in cystic fibrosis Armstrong, David K; Cunningham, Steve; Davies, Jane C ...
Archives of Disease in Childhood,
05/2014, Volume:
99, Issue:
5
Journal Article, Book Review
Peer reviewed
The principal cause of morbidity and mortality in cystic fibrosis (CF) is pulmonary disease, so the focus of new treatments in this condition is primarily targeted at the lungs. Since the cloning of ...the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in 1989, there has been significant interest in the possibility of gene therapy as a treatment for CF. Early studies using viral vectors carrying a healthy CFTR plasmid highlighted the difficulties with overcoming the body's host defences. This article reviews the work on gene therapy in CF to date and describes the ongoing work of the UK CF Gene Therapy Consortium in investigating the potential of gene therapy as a treatment for patients with CF.
•Design, fabrication and validation of a new class of microfluidic pumps and valves.•Precise control of both flow rate and faithful selection of microscale solutions.•Automated, real-time inline ...electrochemical monitoring of cellular microphysiology.•Astrocyte differentiation from hiPSCs for both a control subject and a TSC patient.•First report of altered glutamate metabolism in human astrocytes.
There is a need for valves and pumps that operate at the microscale with precision and accuracy, are versatile in their application, and are easily fabricated. To that end, we developed a new rotary planar multiport valve to faithfully select solutions (contamination = 5.22 ± 0.06 ppb) and a rotary planar peristaltic pump to precisely control fluid delivery (flow rate = 2.4 ± 1.7–890 ± 77 μL/min). Both the valve and pump were implemented in a planar format amenable to single-layer soft lithographic fabrication. These planar microfluidics were actuated by a rotary motor controlled remotely by custom software. Together, these two devices constitute an innovative microformulator that was used to prepare precise, high-fidelity mixtures of up to five solutions (deviation from prescribed mixture = ±|0.02 ± 0.02| %). This system weighed less than a kilogram, occupied around 500 cm3, and generated pressures of 255 ± 47 kPa. This microformulator was then combined with an electrochemical sensor creating a microclinical analyzer (μCA) for detecting glutamate in real time. Using the chamber of the μCA as an in-line bioreactor, we compared glutamate homeostasis in human astrocytes differentiated from human-induced pluripotent stem cells (hiPSCs) from a control subject (CC-3) and a Tuberous Sclerosis Complex (TSC) patient carrying a pathogenic TSC2 mutation. When challenged with glutamate, TSC astrocytes took up less glutamate than control cells. These data validate the analytical power of the μCA and the utility of the microformulator by leveraging it to assess disease-related alterations in cellular homeostasis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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