Throughout the Ebola virus disease (EVD) epidemic in West Africa, field laboratory testing for EVD has relied on complex, multi-step real-time reverse transcription PCR (RT-PCR) assays; an accurate ...sample-to-answer RT-PCR test would reduce time to results and potentially increase access to testing. We evaluated the performance of the Cepheid GeneXpert Ebola assay on clinical venipuncture whole blood (WB) and buccal swab (BS) specimens submitted to a field biocontainment laboratory in Sierra Leone for routine EVD testing by RT-PCR ("Trombley assay").
This study was conducted in the Public Health England EVD diagnostic laboratory in Port Loko, Sierra Leone, using residual diagnostic specimens remaining after clinical testing. EDTA-WB specimens (n = 218) were collected from suspected or confirmed EVD patients between April 1 and July 20, 2015. BS specimens (n = 71) were collected as part of a national postmortem screening program between March 7 and July 20, 2015. EDTA-WB and BS specimens were tested with Xpert (targets: glycoprotein GP and nucleoprotein NP genes) and Trombley (target: NP gene) assays in parallel. All WB specimens were fresh; 84/218 were tested in duplicate on Xpert to compare WB sampling methods (pipette versus swab); 43/71 BS specimens had been previously frozen. In all, 7/218 (3.2%) WB and 7/71 (9.9%) BS samples had Xpert results that were reported as "invalid" or "error" and were excluded, leaving 211 WB and 64 BS samples with valid Trombley and Xpert results. For WB, 22/22 Trombley-positive samples were Xpert-positive (sensitivity 100%, 95% CI 84.6%-100%), and 181/189 Trombley-negative samples were Xpert-negative (specificity 95.8%, 95% confidence interval (CI) 91.8%-98.2%). Seven of the eight Trombley-negative, Xpert-positive (Xpert cycle threshold Ct range 37.7-43.4) WB samples were confirmed to be follow-up submissions from previously Trombley-positive EVD patients, suggesting a revised Xpert specificity of 99.5% (95% CI 97.0%-100%). For Xpert-positive WB samples (n = 22), Xpert NP Ct values were consistently lower than GP Ct values (mean difference -4.06, 95% limits of agreement -6.09, -2.03); Trombley (NP) Ct values closely matched Xpert NP Ct values (mean difference -0.04, 95% limits of agreement -2.93, 2.84). Xpert results (positive/negative) for WB sampled by pipette versus swab were concordant for 78/79 (98.7%) WB samples, with comparable Ct values for positive results. For BS specimens, 20/20 Trombley-positive samples were Xpert-positive (sensitivity 100%, 95% CI 83.2%-100%), and 44/44 Trombley-negative samples were Xpert-negative (specificity 100%, 95% CI 92.0%-100%). This study was limited to testing residual diagnostic samples, some of which had been frozen before use; it was not possible to test the performance of the Xpert Ebola assay at point of care.
The Xpert Ebola assay had excellent performance compared to an established RT-PCR benchmark on WB and BS samples in a field laboratory setting. Future studies should evaluate feasibility and performance outside of a biocontainment laboratory setting to facilitate expanded access to testing.
Despite wide use in clinical practice, acupuncture remains a controversial treatment for chronic pain. Our objective was to update an individual patient data meta-analysis to determine the effect ...size of acupuncture for 4 chronic pain conditions. We searched MEDLINE and the Cochrane Central Registry of Controlled Trials randomized trials published up until December 31, 2015. We included randomized trials of acupuncture needling versus either sham acupuncture or no acupuncture control for nonspecific musculoskeletal pain, osteoarthritis, chronic headache, or shoulder pain. Trials were only included if allocation concealment was unambiguously determined to be adequate. Raw data were obtained from study authors and entered into an individual patient data meta-analysis. The main outcome measures were pain and function. An additional 13 trials were identified, with data received for a total of 20,827 patients from 39 trials. Acupuncture was superior to sham as well as no acupuncture control for each pain condition (all P < .001) with differences between groups close to .5 SDs compared with no acupuncture control and close to .2 SDs compared with sham. We also found clear evidence that the effects of acupuncture persist over time with only a small decrease, approximately 15%, in treatment effect at 1 year. In secondary analyses, we found no obvious association between trial outcome and characteristics of acupuncture treatment, but effect sizes of acupuncture were associated with the type of control group, with smaller effects sizes for sham controlled trials that used a penetrating needle for sham, and for trials that had high intensity of intervention in the control arm. We conclude that acupuncture is effective for the treatment of chronic pain, with treatment effects persisting over time. Although factors in addition to the specific effects of needling at correct acupuncture point locations are important contributors to the treatment effect, decreases in pain after acupuncture cannot be explained solely in terms of placebo effects. Variations in the effect size of acupuncture in different trials are driven predominantly by differences in treatments received by the control group rather than by differences in the characteristics of acupuncture treatment.
Acupuncture is effective for the treatment of chronic musculoskeletal, headache, and osteoarthritis pain. Treatment effects of acupuncture persist over time and cannot be explained solely in terms of placebo effects. Referral for a course of acupuncture treatment is a reasonable option for a patient with chronic pain.
There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome ...provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B
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The quantum chromodynamics (QCD) axion may modify the cooling rates of neutron stars (NSs). The axions are produced within the NS cores from nucleon bremsstrahlung and, when the nucleons are in ...superfluid states, Cooper pair breaking and formation processes. We show that four of the nearby isolated magnificent seven NSs along with PSR J0659 are prime candidates for axion cooling studies because they are coeval, with ages of a few hundred thousand years known from kinematic considerations, and they have well-measured surface luminosities. We compare these data to dedicated NS cooling simulations incorporating axions, profiling over uncertainties related to the equation of state, NS masses, surface compositions, and superfluidity. Our calculations of the axion and neutrino emissivities include high-density suppression factors that also affect SN 1987A and previous NS cooling limits on axions. We find no evidence for axions in the isolated NS data, and within the context of the Kim-Shifman-Vainshtein-Zakharov QCD axion model, we constrain m_{a}≲16 meV at 95% confidence level. An improved understanding of NS cooling and nucleon superfluidity could further improve these limits or lead to the discovery of the axion at weaker couplings.
Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined ...the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of BCR-ABL1 (n = 46) or ETV6-RUNX1 (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of those with CRLF2 rearrangement). Among the remaining patients, using reverse transcriptase polymerase chain reaction or transcriptome sequencing, we identified targetable ABL-class fusions (ABL1, ABL2, CSF1R, and PDGFRB) in 14.1%, EPOR rearrangements or JAK2 fusions in 8.8%, alterations activating other JAK-STAT signaling genes (IL7R, SH2B3, JAK1) in 6.3% or other kinases (FLT3, NTRK3, LYN) in 4.6%, and mutations involving the Ras pathway (KRAS, NRAS, NF1, PTPN11) in 6% of those with Ph-like ALL. We identified 8 new rearrangement partners for 4 kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL implemented in Children's Oncology Group ALL trials.
•Ph-like ALL is characterized by a diverse array of genetic alterations activating cytokine receptor and tyrosine kinase signaling.•Pediatric patients with Ph-like ALL can be identified in real time for effective treatment stratification.
Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis ...of the genome of Euprymna scolopes, a model cephalopod with richly characterized host–microbe interactions. We identified largescale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host–symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes. Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.
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► ZSM-5 for glucose, furan, and maple wood catalytic fast pyrolysis. ► Maximum aromatic yield from glucose pyrolysis on HZSM-5 at SiO2/Al2O3 ratio=30. ► Mesoporous HZSM-5 favors ...production of larger aromatics. ► Removing external surface acid sites does not affect product distribution.
The conversion of glucose, furan and maple wood has been investigated over different types of ZSM-5 catalyst in semi-batch and fixed-bed reactors. The aromatic yield from glucose conversion goes through a maximum as a function of the framework silica-to-alumina ratio (SAR) of ZSM-5 with an optimum at SAR=30. This suggests that the concentration of acid sites inside the zeolite is critical for maximizing aromatic yield. Creating hierarchical mesopores within the zeolite slightly increased of coke formation and decreased the formation of the monocyclic aromatics. Mesoporous ZSM-5 was also observed to favor the production of larger alkylated monoaromatics. The selective removal of external acid sites from the ZSM-5 catalysts only slightly increased the catalyst activity but also decreased the selectivity to the desired aromatic products.
Summary
The rice blast fungus Magnaporthe oryzae elaborates a specialized cell called an appressorium, which is used to breach the tough outer cuticle of a rice leaf, enabling the fungus entry to ...host plant cells. The appressorium generates enormous turgor by accumulating glycerol to very high concentrations within the cell. Glycerol accumulation and melanization of the appressorium cell wall collectively drive turgor‐mediated penetration of the rice leaf. In this review, we discuss the potential metabolic sources of glycerol in the rice blast fungus and how appressorium turgor is focused as physical force at the base of the infection cell, leading to the formation of a rigid penetration peg. We review recent studies of M. oryzae and other relevant appressorium‐forming fungi which shed light on how glycerol is synthesized and how appressorium turgor is regulated. Finally, we provide some questions to guide avenues of future research that will be important in fully understanding the role of glycerol in rice blast disease.
The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing ...in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.
The adsorption of biomolecules to the surface of engineered nanomaterials, known as corona formation, defines their biological identity by altering their surface properties and transforming the ...physical, chemical and biological characteristics of the particles. In the first decade since the term protein corona was coined, studies have focused primarily on biomedical applications and human toxicity. The relevance of the environmental dimensions of the protein corona is still emerging. Often referred to as the eco-corona, a biomolecular coating forms upon nanomaterials as they enter the environment and may include proteins, as well as a diverse array of other biomolecules such as metabolites from cellular activity and/or natural organic matter. Proteins remain central in studies of eco-coronas because of the ease of monitoring and structurally characterizing proteins, as well as their crucial role in receptor engagement and signalling. The proteins within the eco-corona are optimal targets to establish the biophysicochemical principles of corona formation and transformation, as well as downstream impacts on nanomaterial uptake, distribution and impacts on the environment. Moreover, proteins appear to impart a biological identity, leading to cellular or organismal recognition of nanomaterials, a unique characteristic compared with natural organic matter. We contrast insights into protein corona formation from clinical samples with those in environmentally relevant systems. Principles specific to the environment are also explored to gain insights into the dynamics of interaction with or replacement by other biomolecules, including changes during trophic transfer and ecotoxicity. With many challenges remaining, we also highlight key opportunities for method development and impactful systems on which to focus the next phase of eco-corona studies. By interrogating these environmental dimensions of the protein corona, we offer a perspective on how mechanistic insights into protein coronas in the environment can lead to more sustainable, environmentally safe nanomaterials, as well as enhancing the efficacy of nanomaterials used in remediation and in the agri-food sector.
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