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Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80–90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.

Background Postoperative deterioration is often preceded by abnormal vital parameters. Therefore, vital parameters of postoperative patients are routinely measured by nursing staff. Wrist-worn sensors could potentially provide an alternative tool for the measurement of vital parameters in low-acuity settings. These devices would allow more frequent or even continuous measurements of vital parameters without relying on time-consuming manual measurements, provided their accuracy in this clinical population is established. Objective This study aimed to assess the accuracy of heart rate (HR) and respiratory rate (RR) measures obtained via a wearable photoplethysmography (PPG) wristband in a cohort of postoperative patients. Methods The accuracy of the wrist-worn PPG sensor was assessed in 62 post–abdominal surgery patients (mean age 55, SD 15 years; median BMI 34, IQR 25-40 kg/m2). The wearable obtained HR and RR measurements were compared to those of the reference monitor in the postanesthesia or intensive care unit. Bland-Altman and Clarke error grid analyses were performed to determine agreement and clinical accuracy. Results Data were collected for a median of 1.2 hours per patient. With a coverage of 94% for HR and 34% for RR, the device was able to provide accurate measurements for the large majority of the measurements as 98% and 93% of the measurements were within 5 bpm or 3 rpm of the reference signal. Additionally, 100% of the HR and 98% of the RR measurements were clinically acceptable on Clarke error grid analysis. Conclusions The wrist-worn PPG device is able to provide measurements of HR and RR that can be seen as sufficiently accurate for clinical applications. Considering the coverage, the device was able to continuously monitor HR and report RR when measurements of sufficient quality were obtained. Trial Registration ClinicalTrials.gov NCT03923127; https://www.clinicaltrials.gov/ct2/show/NCT03923127

We present the analysis of a spectroscopic secondary eclipse of the hottest transiting exoplanet detected to date, KELT-9b, obtained with the Wide Field Camera 3 aboard the Hubble Space Telescope. We complement these data with literature information on stellar pulsations and Spitzer/Infrared Array Camera and Transiting Exoplanet Survey Satellite eclipse depths of this target to obtain a broadband thermal emission spectrum. Our extracted spectrum exhibits a clear turnoff at 1.4$\mu$m. This points to H$^{-}$ bound-free opacities shaping the spectrum. To interpret the spectrum, we perform grid retrievals of self-consistent 1D equilibrium chemistry forward models, varying the composition and energy budget. The model with solar metallicity and C/O ratio provides a poor fit because the H$^{-}$ signal is stronger than expected, requiring an excess of electrons. This pushes our retrievals toward high atmospheric metallicities ($[M/H]=1.98^{+0.19}_{-0.21}$) and a C/O ratio that is subsolar by 2.4$\sigma$. We question the viability of forming such a high-metallicity planet, and therefore provide other scenarios to increase the electron density in this atmosphere. We also look at an alternative model in which we quench TiO and VO. This fit results in an atmosphere with a slightly subsolar metallicity and subsolar C/O ratio ($[M/H]=-0.22^{+0.17}_{-0.13}$, log(C/O)$=-0.34^{+0.19}_{-0.34}$). However, the required TiO abundances are disputed by recent high-resolution measurements of the same planet., Comment: 8 pages, 4 figures, Accepted for publication in A&A letters

Gamma-aminobutyric acid (GABA) and glutamate are principal neurotransmitters essential for late gestational brain development and may play an important role in prematurity-related brain injury. In vivo investigation of GABA in the preterm infant with standard proton magnetic resonance spectroscopy (1H-MRS) has been limited due to its low concentrations in the developing brain, and overlap in the spectrum by other dominant metabolites. We describe early postnatal profiles of in vivo GABA and glutamate concentrations in the developing preterm brain measured by using the J-difference editing technique, Mescher-Garwood point resolved spectroscopy. We prospectively enrolled very preterm infants born ≤32 weeks gestational age and non-sedated 1H-MRS (echo time 68 ms, relaxation time 2000 ms, 256 signal averages) was acquired on a 3 Tesla magnetic resonance imaging scanner from a right frontal lobe voxel. Concentrations of GABA + (with macromolecules) was measured from the J-difference spectra; whereas glutamate and composite glutamate + glutamine (Glx) were measured from the unedited (OFF) spectra and reported in institutional units. We acquired 42 reliable spectra from 38 preterm infants without structural brain injury [median gestational age at birth of 28.0 (IQR 26.0, 28.9) weeks; 19 males (50%)] at a median postmenstrual age of 38.4 (range 33.4 to 46.4) weeks. With advancing post-menstrual age, the concentrations of glutamate OFF increased significantly, adjusted for co-variates (generalized estimating equation β = 0.22, p = 0.02). Advancing postnatal weeks of life at the time of imaging positively correlated with GABA + (β = 0.06, p = 0.02), glutamate OFF (β = 0.11, p = 0.02) and Glx OFF (β = 0.12, p = 0.04). Male infants had higher GABA + (1.66 ± 0.07 vs. 1.33 ± 0.11, p = 0.01) concentrations compared with female infants. For the first time, we report the early ex-utero developmental profile of in vivo GABA and glutamate stratified by age and sex in the developing brain of very preterm infants. This data may provide novel insights into the pathophysiology of neurodevelopmental disabilities reported in preterm infants even in the absence of structural brain injury.

The warming and subsequent degradation of mountain permafrost within alpine areas represent an important process influencing the stability of steep slopes and rock faces. The unstable and monitored slopes of Mannen (Møre and Romsdal county, southern Norway) and Gámanjunni-3 (Troms and Finnmark county, northern Norway) were classified as high-risk sites by the Norwegian Geological Survey (NGU). Failure initiation has been suggested to be linked to permafrost degradation, but the detailed permafrost distribution at the sites is unknown. Rock wall (RW) temperature loggers at both sites have measured the thermal regime since 2015, showing mean rock surface temperatures between 2.5 and −1.6 ∘C depending on site and topographic aspect. Between 2016 and 2019 we conducted 2D and 3D electrical resistivity tomography (ERT) surveys on the plateau and directly within the rock wall back scarp of the unstable slopes at both sites. In combination with geophysical laboratory analysis of rock wall samples from both sites, the ERT soundings indicate widespread permafrost areas, especially at Gámanjunni-3. Finally, we conducted 2D thermal modelling to evaluate the potential thermal regime, along with an analysis of available displacement rate measurements based on Global Navigation Satellite System (GNSS) and ground- and satellite-based interferometric synthetic aperture radar (InSAR) methods. Surface air and ground temperatures have increased significantly since ca. 1900 by 1 and 1.5 ∘C, and the highest temperatures have been measured and modelled since 2000 at both study sites. We observed a seasonality of displacement, with increasing velocities during late winter and early spring and the highest velocities in June, probably related to water pressure variations during snowmelt. The displacement rates of Gámanjunni-3 rockslide co-vary with subsurface resistivity and modelled ground temperature. Increased displacement rates seem to be associated with sub-zero ground temperatures and higher ground resistivity. This might be related to the presence of ground ice in fractures and pores close to the melting point, facilitating increased deformation. The study demonstrates and discusses the possible influence of permafrost, at least locally, on the dynamics of large rock slope instabilities.

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called “oligo stitching”. Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitching’s versatility and high efficiency, we expect that it will be a useful addition to the molecular biologist’s toolbox.Abstract Graphic

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the Fe(II) active site to form a highly reactive Fe(IV) = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-Fe(IV) = O intermediate and characterized its structure as a Fe(II)-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate–bound ternary Fe(II) active site before the O(2) reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of Fe(IV) = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the Fe(II) site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the Fe(II) site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.

The surface topography of diamond coatings strongly affects surface properties such as adhesion, friction, wear, and biocompatibility. However, the understanding of multi-scale topography, and its effect on properties, has been hindered by conventional measurement methods, which capture only a single length scale. Here, four different polycrystalline diamond coatings are characterized using transmission electron microscopy to assess the roughness down to the sub-nanometer scale. Then these measurements are combined, using the power spectral density (PSD), with conventional methods (stylus profilometry and atomic force microscopy) to characterize all scales of topography. The results demonstrate the critical importance of measuring topography across all length scales, especially because their PSDs cross over one another, such that a surface that is rougher at a larger scale may be smoother at a smaller scale and vice versa. Furthermore, these measurements reveal the connection between multi-scale topography and grain size, with characteristic scaling behavior at and slightly below the mean grain size, and self-affine fractal-like roughness at other length scales. At small (subgrain) scales, unpolished surfaces exhibit a common form of residual roughness that is self-affine in nature but difficult to detect with conventional methods. This approach of capturing topography from the atomic- to the macro-scale is termed comprehensive topography characterization, and all of the topography data from these surfaces has been made available for further analysis by experimentalists and theoreticians. Scientifically, this investigation has identified four characteristic regions of topography scaling in polycrystalline diamond materials., 13 pages, 6 figures

This study intends to set up computational fluid dynamic simulations along with static and dynamic stress simulations to determine maximum possible accelerations and stress levels that may occur for poles having a single luminaire under certain typical and extreme environmental conditions.