The kappa coefficient is not an index of accuracy, indeed it is not an index of overall agreement but one of agreement beyond chance. Chance agreement is, however, irrelevant in an accuracy ...assessment and is anyway inappropriately modelled in the calculation of a kappa coefficient for typical remote sensing applications. The magnitude of a kappa coefficient is also difficult to interpret. Values that span the full range of widely used interpretation scales, indicating a level of agreement that equates to that estimated to arise from chance alone all the way through to almost perfect agreement, can be obtained from classifications that satisfy demanding accuracy targets (e.g. for a classification with overall accuracy of 95% the range of possible values of the kappa coefficient is −0.026 to 0.900). Comparisons of kappa coefficients are particularly challenging if the classes vary in their abundance (i.e. prevalence) as the magnitude of a kappa coefficient reflects not only agreement in labelling but also properties of the populations under study. It is shown that all of the arguments put forward for the use of the kappa coefficient in accuracy assessment are flawed and/or irrelevant as they apply equally to other, sometimes easier to calculate, measures of accuracy. Calls for the kappa coefficient to be abandoned from accuracy assessments should finally be heeded and researchers are encouraged to provide a set of simple measures and associated outputs such as estimates of per-class accuracy and the confusion matrix when assessing and comparing classification accuracy.
•Kappa is not a measure of accuracy but of agreement beyond chance and chance correction is not needed.•All arguments for the use of kappa are flawed or apply equally to other measures.•Interpreting a kappa coefficient is difficult due especially to the effects of prevalence and bias.•A very accurate classification could be associated with a very wide range of kappa values.•Kappa should not be routinely used in accuracy assessment or comparison.
We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair ...to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.
has long been a premier model for the development and application of cutting-edge genetic approaches. The CRISPR-Cas system now adds the ability to manipulate the genome with ease and precision, ...providing a rich toolbox to interrogate relationships between genotype and phenotype, to delineate and visualize how the genome is organized, to illuminate and manipulate RNA, and to pioneer new gene drive technologies. Myriad transformative approaches have already originated from the CRISPR-Cas system, which will likely continue to spark the creation of tools with diverse applications. Here, we provide an overview of how CRISPR-Cas gene editing has revolutionized genetic analysis in
and highlight key areas for future advances.
We present the fourth in a series of catalogs of gamma-ray bursts (GRBs) observed with Fermi's Gamma-ray Burst Monitor (Fermi-GBM). It extends the six year catalog by four more years, now covering ...the 10 year time period from trigger enabling on 2008 July 12 to 2018 July 11. During this time period GBM triggered almost twice a day on transient events, 2356 of which we identified as cosmic GRBs. Additional trigger events were due to solar flare events, magnetar burst activities, and terrestrial gamma-ray flashes. The intention of the GBM GRB catalog series is to provide updated information to the community on the most important observables of the GBM-detected GRBs. For each GRB the location and main characteristics of the prompt emission, the duration, peak flux, and fluence are derived. The latter two quantities are calculated for the 50-300 keV energy band, where the maximum energy release of GRBs in the instrument reference system is observed and also for a broader energy band from 10-1000 keV, exploiting the full energy range of GBM's low-energy detectors. Furthermore, information is given on the settings of the triggering criteria and exceptional operational conditions during years 7 to 10 in the mission. This fourth catalog is an official product of the Fermi-GBM science team, and the data files containing the complete results are available from the High-Energy Astrophysics Science Archive Research Center.
We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. ...This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
Sulfur and selenium occur in proteins as constituents of the amino acids cysteine, methionine, selenocysteine, and selenomethionine. Recent research underscores that these amino acids are truly ...exceptional. Their redox activity under physiological conditions allows an amazing variety of posttranslational protein modifications, metal free redox pathways, and unusual chalcogen redox states that increasingly attract the attention of biological chemists. Unlike any other amino acid, the “redox chameleon” cysteine can participate in several distinct redox pathways, including exchange and radical reactions, as well as atom‐, electron‐, and hydride‐transfer reactions. It occurs in various oxidation states in the human body, each of which exhibits distinctive chemical properties (e.g. redox activity, metal binding) and biological activity. The position of selenium in the periodic table between the metals and the nonmetals makes selenoproteins ideal catalysts for many biological redox transformations. It is therefore apparent that the chalcogen amino acids cysteine, methionine, selenocysteine, and selenomethionine exhibit a unique biological chemistry that is the source of exciting research opportunities.
Biochemical chameleon: Chalcogen‐containing amino acids play an important role in biological redox chemistry. The oxidation state of sulfur in cysteine varies from −2 to +6, and therefore cysteine can participate in a wide range of redox reactions, such as atom‐, electron‐, and hydride transfer, as well as radical and exchange reactions (see scheme). Mechanistic, structural, and functional aspects of sulfur‐ and selenium‐containing proteins are discussed.
The intestine secretes a range of hormones with important local and distant actions, including the control of insulin secretion and appetite. A number of enteroendocrine cell types have been ...described, each characterized by a distinct hormonal signature, such as K-cells producing glucose-dependent insulinotropic polypeptide (GIP), L-cells producing glucagon-like peptide-1 (GLP-1), and I-cells producing cholecystokinin (CCK). To evaluate similarities between L-, K-, and other enteroendocrine cells, primary murine L- and K-cells, and pancreatic α- and β-cells, were purified and analyzed by flow cytometry and microarray-based transcriptomics. By microarray expression profiling, L cells from the upper small intestinal (SI) more closely resembled upper SI K-cells than colonic L-cells. Upper SI L-cell populations expressed message for hormones classically localized to different enteroendocrine cell types, including GIP, CCK, secretin, and neurotensin. By immunostaining and fluorescence-activated cell sorting analysis, most colonic L-cells contained GLP-1 and PeptideYY In the upper SI, most L-cells contained CCK, approximately 10% were GIP positive, and about 20% were PeptideYY positive. Upper SI K-cells exhibited approximately 10% overlap with GLP-1 and 6% overlap with somatostatin. Enteroendocrine-specific transcription factors were identified from the microarrays, of which very few differed between the enteroendocrine cell populations. Etv1, Prox1, and Pax4 were significantly enriched in L-cells vs. K cells by quantitative RT-PCR. In summary, our data indicate a strong overlap between upper SI L-, K-, and I-cells and suggest they may rather comprise a single cell type, within which individual cells exhibit a hormonal spectrum that may reflect factors such as location along the intestine and exposure to dietary nutrients.
The Aerosol Robotic Network (AERONET) Version 3 (V3) aerosol retrieval algorithm is described, which is based on the Version 2 (V2)
algorithm with numerous updates. Comparisons of V3 aerosol ...retrievals to
those of V2 are presented, along with a new approach to estimate
uncertainties in many of the retrieved aerosol parameters. Changes in the V3 aerosol retrieval algorithm include (1) a new polarized radiative transfer code (RTC), which replaced the scalar RTC of V2, (2) detailed
characterization of gas absorption by adding NO2 and H2O to
specify total gas absorption in the atmospheric column, specification of
vertical profiles of all the atmospheric species, (3) new bidirectional reflectance distribution function (BRDF) parameters for land sites adopted
from the MODIS BRDF/Albedo product, (4) a new version of the extraterrestrial
solar flux spectrum, and (5) a new temperature correction procedure of both direct Sun and sky radiance measurements. The potential effect of each change in V3 on single scattering albedo (SSA) retrievals was analyzed. The
operational almucantar retrievals of V2 versus V3 were compared for four AERONET sites: GSFC, Mezaira, Mongu, and Kanpur. Analysis showed very good
agreement in retrieved parameters of the size distributions. Comparisons of
SSA retrievals for dust aerosols (Mezaira) showed a good agreement in 440 nm
SSA, while for longer wavelengths V3 SSAs are systematically higher than those of V2, with the largest mean difference at 675 nm due to cumulative
effects of both extraterrestrial solar flux and BRDF changes. For non-dust
aerosols, the largest SSA deviation is at 675 nm due to differences in
extraterrestrial solar flux spectrums used in each version. Further, the SSA
675 nm mean differences are very different for weakly (GSFC) and strongly
(Mongu) absorbing aerosols, which is explained by the lower sensitivity to a bias in aerosol scattering optical depth by less absorbing aerosols. A new
hybrid (HYB) sky radiance measurement scan is introduced and discussed. The HYB combines features of scans in two different planes to maximize the range
of scattering angles and achieve scan symmetry, thereby allowing for cloud
screening and spatial averaging, which is an advantage over the principal plane scan that lacks robust symmetry. We show that due to an extended range of scattering angles, HYB SSA retrievals for dust aerosols exhibit smaller
variability with solar zenith angles (SZAs) than those of almucantar (ALM), which allows extension of HYB SSA retrievals to SZAs less than 50∘ to as small as 25∘. The comparison of SSA retrievals from closely
time-matched HYB and ALM scans in the 50 to 75∘ SZA range showed good agreement with the differences below ∼0.005. We also present an approach to estimate retrieval uncertainties which
utilizes the variability in retrieved parameters generated by perturbing
both measurements and auxiliary input parameters as a proxy for retrieval uncertainty. The perturbations in measurements and auxiliary inputs are
assumed as estimated biases in aerosol optical depth (AOD), radiometric
calibration of sky radiances combined with solar spectral irradiance, and
surface reflectance. For each set of Level 2 Sun/sky radiometer
observations, 27 inputs corresponding to 27 combinations of biases were
produced and separately inverted to generate the following statistics of
the inversion results: average, standard deviation, minimum and maximum
values. From these statistics, standard deviation (labeled U27) is used as a proxy for estimated uncertainty, and a lookup table (LUT) approach was implemented to reduce the computational time. The U27 climatological LUT was
generated from the entire AERONET almucantar (1993–2018) and hybrid
(2014–2018) scan databases by binning U27s in AOD (440 nm), Angström exponent (AE, 440–870 nm), and SSA (440, 675, 870, 1020 nm). Using this LUT approach, the uncertainty estimates U27 for each individual V3 Level 2 retrieval can
be obtained by interpolation using the corresponding measured and inverted
combination of AOD, AE, and SSA.
Aims/hypothesis
Intra-islet and gut–islet crosstalk are critical in orchestrating basal and postprandial metabolism. The aim of this study was to identify regulatory proteins and receptors underlying ...somatostatin secretion though the use of transcriptomic comparison of purified murine alpha, beta and delta cells.
Methods
Sst-Cre
mice crossed with fluorescent reporters were used to identify delta cells, while
Glu-Venus
(with Venus reported under the control of the
Glu
also known as
Gcg
promoter) mice were used to identify alpha and beta cells. Alpha, beta and delta cells were purified using flow cytometry and analysed by RNA sequencing. The role of the ghrelin receptor was validated by imaging delta cell calcium concentrations using islets with delta cell restricted expression of the calcium reporter GCaMP3, and in perfused mouse pancreases.
Results
A database was constructed of all genes expressed in alpha, beta and delta cells. The gene encoding the ghrelin receptor,
Ghsr
, was highlighted as being highly expressed and enriched in delta cells. Activation of the ghrelin receptor raised cytosolic calcium levels in primary pancreatic delta cells and enhanced somatostatin secretion in perfused pancreases, correlating with a decrease in insulin and glucagon release. The inhibition of insulin secretion by ghrelin was prevented by somatostatin receptor antagonism.
Conclusions/interpretation
Our transcriptomic database of genes expressed in the principal islet cell populations will facilitate rational drug design to target specific islet cell types. The present study indicates that ghrelin acts specifically on delta cells within pancreatic islets to elicit somatostatin secretion, which in turn inhibits insulin and glucagon release. This highlights a potential role for ghrelin in the control of glucose metabolism.
On 2017 August 17 at 12:41:06 UTC the Fermi Gamma-ray Burst Monitor (GBM) detected and triggered on the short gamma-ray burst (GRB) 170817A. Approximately 1.7 s prior to this GRB, the Laser ...Interferometer Gravitational-wave Observatory triggered on a binary compact merger candidate associated with the GRB. This is the first unambiguous coincident observation of gravitational waves and electromagnetic radiation from a single astrophysical source and marks the start of gravitational-wave multi-messenger astronomy. We report the GBM observations and analysis of this ordinary short GRB, which extraordinarily confirms that at least some short GRBs are produced by binary compact mergers.
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