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Continuous water vapour measurements in the troposphere and lowermost stratosphere are crucial for the understanding of global water transport processes and climate impact. In the course of the ‘Development and Evaluation of Novel and Compact Hygrometer for Airborne Research’ (DENCHAR) project, water vapour instruments (WaSuL, WVSS‑II, ICH) have been partly newly developed and/or extensively tested in the laboratory as well as onboard of research aircraft. For the blind intercomparisons of the instruments, an MBW DP30 frostpoint mirror and the established FISH Lyman‑α instrument (calibrated versus the MBW DP30) served as reference hygrometers in the laboratory and during the flights, respectively. All DENCHAR-instruments show very consistent behaviour in their respective measuring ranges with deviations of less than about 5–10 %, in‑flight with respect to FISH and in the laboratory with respect to DP30 in the range between 100 and 1000 ppmv, as well as among each other in the range of 100 to 20000 ppmv. At mixing ratios below 100 ppmv, differences between the instruments appear which depend on the individual response times and calibrations. In summary, the WaSul, ICH and WVSS‑II hygrometers can be recommended for continuous water vapour measurements at mixing ratios larger than 10, 30, and 30 ppmv, respectively. For an accurate, reliable, and stable measurement of lower water vapour mixing ratios with a compact hygrometer suitable for autonomous operation, either these instruments need to be improved or new technology would have to be developed. In addition to the instrument evaluation, the performance of different water vapour inlet systems is addressed by comparing a forward-facing Rosemount (TAT housing) and a wall plate inlet. From all in‑flight tests, no measurement artifacts caused by specific characteristics of an inlet could be identified, i.e., forward-facing Rosemount and wall plate inlets can be recommended for use on aircraft for water vapour mixing ratios above 30 ppmv.

We present the results of the human genomic small variant calling benchmarking initiative of the German Research Foundation (DFG) funded Next Generation Sequencing Competence Network (NGS-CN) and the German Human Genome-Phenome Archive (GHGA). In this effort, we developed NCBench, a continuous benchmarking platform for the evaluation of small genomic variant callsets in terms of recall, precision, and false positive/negative error patterns. NCBench is implemented as a continuously re-evaluated open-source repository. We show that it is possible to entirely rely on public free infrastructure (Github, Github Actions, Zenodo) in combination with established open-source tools. NCBench is agnostic of the used dataset and can evaluate an arbitrary number of given callsets, while reporting the results in a visual and interactive way. We used NCBench to evaluate over 40 callsets generated by various variant calling pipelines available in the participating groups that were run on three exome datasets from different enrichment kits and at different coverages. While all pipelines achieve high overall quality, subtle systematic differences between callers and datasets exist and are made apparent by NCBench.These insights are useful to improve existing pipelines and develop new workflows. NCBench is meant to be open for the contribution of any given callset. Most importantly, for authors, it will enable the omission of repeated re-implementation of paper-specific variant calling benchmarks for the publication of new tools or pipelines, while readers will benefit from being able to (continuously) observe the performance of tools and pipelines at the time of reading instead of at the time of writing.

Research infrastructures play an increasingly essential role in scientific research. They provide rich data sources for scientists, such as services and software packages, via catalog and virtual research environments. However, such research infrastructures are typically domain-specific and often not connected. Accordingly, researchers and practitioners face fundamental challenges introduced by fragmented knowledge from heterogeneous, autonomous sources with complicated and uncertain relations in particular research domains. Additionally, the exponential growth rate of knowledge in a specific domain surpasses human experts’ ability to formalize and capture tacit and explicit knowledge efficiently. Thus, a knowledge management system is required to discover knowledge effectively, automate the knowledge acquisition based on artificial intelligence approaches, integrate the captured knowledge, and deliver consistent knowledge to agents, research communities, and end-users. In this study, we present the development process of a knowledge management system for ENVironmental Research Infrastructures, which are crucial pillars for environmental scientists in their quest for understanding and interpreting the complex Earth System. Furthermore, we report the challenges we have faced and discuss the lessons learned during the development process.

The mechanisms by which cells determine their position within the 3D space are poorly understood. Research in salamanders offers fresh insights into this question, uncovering Tig1 as a central determinant of proximo-distal identity in regeneration.

Targeting a gene with two loxP sites is both time and labour intensive. Here the authors present Cre-Controlled CRISPR allowing conditional mutagenesis of a gene of interest and simultaneously labelling the putative mutant cells fluorescently.

Glioblastoma (GBM) is the most common and malignant brain tumor in adults. Genomic and epigenomic alterations of multiple cancer-driving genes are frequent in GBM. To identify molecular alterations associated with epigenetic aberrations, we performed whole exome sequencing-based analysis of DNA copy number variations in 55 adult patients with IDH-wild-type GBM. Beside mutations in common GBM driver genes such as TERTp (76%), TP53 (22%) and PTEN (20%), 67% of patients were affected by amplifications of genes associated with RTK/Rb/p53 cell signaling, including EGFR (45%), CDK4 (13%), and MDM2/4 (both 7%). The minimal deleted region at chromosome 10 was detected at the DNA demethylase TET1 (93%), mainly due to a loss-of-heterozygosity of complete chromosome 10 (53%) or by a mono-allelic microdeletion at 10q21.3 (7%). In addition, bi-allelic TET1 deletions, detected in 18 patients (33%), frequently co-occurred with EGFR amplification and were associated with low levels of TET1 mRNA expression, pointing at loss of TET1 activity. Bi-allelic TET1 loss was not associated with global concentrations of 5-hydroxymethylcytosine, indicating a site-specific effect of TET1 for DNA (de)methylation. Focal amplification of EGFR positively correlated with overall mutational burden, tumor size, and poor long-term survival. Bi-allelic TET1 loss was not an independent prognostic factor, but significantly associated with poor survival in patients with concomitant EGFR amplification. Rates of genomic TET1 deletion were significantly lower in a cohort of IDH1-mutated patients. Despite the relevance of TET1 for DNA demethylation and as potential therapeutic target, a frequent genomic loss of TET1 has not previously been reported in GBM.

The influence of shipping on air quality over the Southern Baltic Sea was investigated by characterizing the horizontal and vertical distribution of aerosols and trace gases using airborne measurements in the summer of 2015. Generally, continental and anthropogenic emissions affected the vertical distribution of atmospheric pollutants, leading to pronounced stratification in and above the marine boundary layer and controlling the aerosol extinction. Marine traffic along the shipping corridor “Kadet Fairway” in the Arkona Basin is shown to influence the presence and properties of both trace gases and aerosol particles in the lowest atmospheric layer. Total particle number concentration and NOy mixing ratio increased in the corridor plumes, relative to background, by a factor 1.55 and 3.45, respectively. Titration, triggered by the enhanced presence of nitrogen compounds, led to a median ozone depletion of 19% in the corridor plumes. The enforcement of the Sulphur Emission Control Area (SECA) might be responsible for the minor sulphur dioxide increase (20%) in the corridor plumes. Ship traffic caused a minor enhancement of black carbon mass concentration, estimated to be around 10%. The study of individual ship plumes indicated that ship emitted aerosol was substantially different from background aerosol: fresh ship exhaust was preferentially enriched in aerosol particles with diameters below 100 nm and in black carbon particles with core diameters above 300-400 nm. With the present work the impact of marine traffic on the concentration and properties of atmospheric components within the marine boundary layer over the open water of the Southern Baltic Sea is assessed with airborne observations for the first time. Due to the high uncertainty affecting the estimations of ship emissions, this dataset represents a valuable reference for the assessment of ship emission inventories and related environmental-climatic impacts on the Southern Baltic Sea.

Recent findings suggest that reduced neurogenesis could be one of the underlying reasons for the exacerbated neuropathology in humans, thus restoring the neural stem cell proliferation and neurogenesis could help to circumvent some pathological aspects of Alzheimer’s disease. We recently identified Interleukin-4/STAT6 signaling as a neuron–glia crosstalk mechanism that enables glial proliferation and neurogenesis in adult zebrafish brain and 3D cultures of human astroglia, which manifest neurogenic properties. In this study, by using single cell sequencing in the APP/PS1dE9 mouse model of AD, we found that IL4 receptor (Il4r) is not expressed in mouse astroglia and IL4 signaling is not active in these cells. We tested whether activating IL4/STAT6 signaling would enhance cell proliferation and neurogenesis in healthy and disease conditions. Lentivirus-mediated expression of IL4R or constitutively active STAT6VT impaired the survival capacity of mouse astroglia in vivo but not in vitro. These results suggest that the adult mouse brain generates a non-permissive environment that dictates a negative effect of IL4 signaling on astroglial survival and neurogenic properties in contrast to zebrafish brains and in vitro mammalian cell cultures. Our findings that IL4R signaling in dentate gyrus (DG) of adult mouse brain impinges on the survival of DG cells implicate an evolutionary mechanism that might underlie the loss of neuroregenerative ability of the brain, which might be utilized for basic and clinical aspects for neurodegenerative diseases.

Cis-regulatory elements are important factors for morphological changes. Here, the authors show widespread divergence of limb and eye regulatory elements in limb loss in snakes and eye degeneration in subterranean mammals respectively.

Following the publication of this article [1], the authors reported that the images of Figs. 1, 2 and 3 were published in the incorrect order, whereby they mismatch with their captions.

Abstract Background The short-lived fish Nothobranchius furzeri is the shortest-lived vertebrate that can be cultured in captivity and was recently established as a model organism for aging research. Small non-coding RNAs, especially miRNAs, are implicated in age dependent control of gene expression. Results Here, we present a comprehensive catalogue of miRNAs and several other non-coding RNA classes (ncRNAs) for Nothobranchius furzeri. Analyzing multiple small RNA-Seq libraries, we show most of these identified miRNAs are expressed in at least one of seven Nothobranchius species. Additionally, duplication and clustering of N. furzeri miRNAs was analyzed and compared to the four fish species Danio rerio, Oryzias latipes, Gasterosteus aculeatus and Takifugu rubripes. A peculiar characteristic of N. furzeri, as compared to other teleosts, was a duplication of the miR-29 cluster. Conclusion The completeness of the catalogue we provide is comparable to that of the zebrafish. This catalogue represents a basis to investigate the role of miRNAs in aging and development in this species.

Adenoviruses (Ads) are large human-pathogenic double-stranded DNA (dsDNA) viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR) and linear-circular homologous recombination (LCHR). We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS). We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS) of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource.

CD8+ T cells are important effectors of adaptive immunity against pathogens, tumors, and self antigens. Here, we asked how human cognate antigen-responsive CD8+ T cells and their receptors could be identified in unselected single-cell gene expression data. Single-cell RNA sequencing and qPCR of dye-labeled antigen-specific cells identified large gene sets that were congruently up- or downregulated in virus-responsive CD8+ T cells under different antigen presentation conditions. Combined expression of TNFRSF9, XCL1, XCL2, and CRTAM was the most distinct marker of virus-responsive cells on a single-cell level. Using transcriptomic data, we developed a machine learning-based classifier that provides sensitive and specific detection of virus-responsive CD8+ T cells from unselected populations. Gene response profiles of CD8+ T cells specific for the autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein differed markedly from virus-specific cells. These findings provide single-cell gene expression parameters for comprehensive identification of rare antigen-responsive cells and T cell receptors.

This work presents the characterisation of the Aerosol Package for the European Research Infrastructure IAGOS (In-service Aircraft for a Global Observing System). Condensation particle counter (CPC) are used to measure the aerosol number concentration of the total and the non-volatile particles. The size distribution is measured by means of an optical particle counter (OPC) in the diameter range of 0.25–3 µm. In particular the OPC is characterised for (1) leakage, (2) flow and calibration stability over the expected IAGOS pressure range of 170 hPa to 1013 hPa, (3) OPC accuracy test by comparing extinction measurements with calculated values from Mie theory using the OPC size distribution and (4) the inter-instrumental precision. The CPC is characterised in the same pressure range for (1) the lower cut-off diameter, (2) the instrument accuracy by comparing with a reference instrument (Faraday Cup Electrometer, FCE) and (3) the instrument precision. We conclude that the IAGOS Aerosol Package is a fully automated, robust, low-maintenance instrument providing high precision measurements with good accuracy.

Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including Cebpa, Gata1, and Klf1. Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.

In 2011, the MOZAIC (Measurement of Ozone by AIRBUS In-Service Aircraft) successor programme IAGOS (In-service Aircraft for a Global Observing System) started to equip their long-haul passenger aircraft with the modified capacitive hygrometer Vaisala HUMICAP® of type H. The assurance of the data quality and the consistency of the data set during the transition from MOZAIC Capacitive Hygrometers to IAGOS Capacitive Hygrometers were evaluated within the CIRRUS-III and AIRTOSS-ICE field studies. During these performance tests, the capacitive hygrometers were operated aboard a Learjet 35A aircraft together with a closed-cell Lyman-α fluorescence hygrometer, an open-path tunable diode laser (TDL) system and a closed-cell, direct TDL absorption hygrometer for water vapour measurement. For MOZAIC-typical operation conditions, the comparison of relative humidity (RH) data from the capacitive hygrometers and reference instruments yielded remarkably good agreement with an uncertainty of 5% RH. The temperature dependence of the sensor's response time was derived from the cross-correlation of capacitive hygrometer data and smoothed data from the fast-responding reference instruments. The resulting exponential moving average function could explain the major part of the observed deviations between the capacitive hygrometers and the reference instruments.

The European Research Infrastructure IAGOS (In-service Aircraft for a Global Observing System) operates a global-scale monitoring system for atmospheric trace gases, aerosols and clouds utilising the existing global civil aircraft. This new monitoring infrastructure builds on the heritage of the former research projects MOZAIC (Measurement of Ozone and Water Vapour on Airbus In-service Aircraft) and CARIBIC (Civil Aircraft for the Regular Investigation of the Atmosphere Based on an Instrument Container). CARIBIC continues within IAGOS and acts as an important airborne measurement reference standard within the wider IAGOS fleet. IAGOS is a major contributor to the in-situ component of the Copernicus Atmosphere Monitoring Service (CAMS), the successor to the Global Monitoring for the Environment and Security – Atmospheric Service, and is providing data for users in science, weather services and atmospherically relevant policy. IAGOS is unique in collecting regular in-situ observations of reactive gases, greenhouse gases and aerosol concentrations in the upper troposphere and lowermost stratosphere (UTLS) at high spatial resolution. It also provides routine vertical profiles of these species in the troposphere over continental sites or regions, many of which are undersampled by other networks or sampling studies, particularly in Africa, Southeast Asia and South America. In combination with MOZAIC and CARIBIC, IAGOS has provided long-term observations of atmospheric chemical composition in the UTLS since 1994. The longest time series are 20 yr of temperature, H2O and O3, and 9–15 yr of aerosols, CO, NO y , CO2, CH4, N2O, SF6, Hg, acetone, ~30 HFCs and ~20 non-methane hydrocarbons. Among the scientific highlights which have emerged from these data sets are observations of extreme concentrations of O3 and CO over the Pacific basin that have never or rarely been recorded over the Atlantic region for the past 12 yr; detailed information on the temporal and regional distributions of O3, CO, H2O, NO y and aerosol particles in the UTLS, including the impacts of cross-tropopause transport, deep convection and lightning on the distribution of these species; characterisation of ice-supersaturated regions in the UTLS; and finally, improved understanding of the spatial distribution of upper tropospheric humidity including the finding that the UTLS is much more humid than previously assumed.

We present the features seen in the first 2 months (July and August 2012) of data collected over the Pacific by IAGOS (In-service Aircraft for a Global Observing System)-equipped aircraft. IAGOS is the continuation and development of the well-known MOZAIC (Measurement of Ozone and Water Vapour on Airbus in-service Aircraft) project where scientific instruments were carried on commercially operated A340 aircraft to make measurements of chemical species in the atmosphere. Here, we show data from an aircraft operated by China Airlines on routes from Taipei to Vancouver, which provided the first trans-Pacific measurements by an IAGOS-equipped aircraft. We describe the chemical composition of the extratropical upper troposphere/lower stratosphere (Ex-UTLS) across the Pacific basin in the Northern Hemisphere. The observed concentrations of ozone span a range from 18 to 500 ppbv indicating sources in the marine boundary layer and lowermost stratosphere, respectively. Concentrations of carbon monoxide (CO) greater than 400 ppbv are observed in the Ex-UTLS suggesting that plumes of pollution have been exported from the continent. These low concentrations of ozone and high concentrations of CO were rarely recorded in 8 yr of MOZAIC observations over the Atlantic.

This article presents the In-service Aircraft of a Global Observing System (IAGOS) developed for operations on commercial long-range Airbus aircraft (A330/A340) for monitoring the atmospheric composition. IAGOS is the continuation of the former Measurement of OZone and water vapour on Airbus In-service airCraft (MOZAIC) programme (1994–2014) with five aircraft operated by European airlines over 20 yr. MOZAIC has provided unique scientific database used worldwide by the scientific community. In continuation of MOZAIC, IAGOS aims to equip a fleet up to 20 aircraft around the world and for operations over decades. IAGOS started in July 2011 with the first instruments installed aboard a Lufthansa A340-300, and a total of six aircraft are already in operation. We present the technical aircraft system concept, with basic instruments for O3, CO, water vapour and clouds; and optional instruments for measuring either NOy, NOx, aerosols or CO2/CH4. In this article, we focus on the O3 and CO instrumentation while other measurements are or will be described in specific papers. O3 and CO are measured by optimised but well-known methods such as UV absorption and IR correlation, respectively. We describe the data processing/validation and the data quality control for O3 and CO. Using the first two overlapping years of MOZAIC/IAGOS, we conclude that IAGOS can be considered as the continuation of MOZAIC with the same data quality of O3 and CO measurements.

We present the results of the human genomic small variant calling benchmarking initiative of the German Research Foundation (DFG) funded Next Generation Sequencing Competence Network (NGS-CN) and the German Human Genome-Phenome Archive (GHGA). In this effort, we developed NCBench, a continuous benchmarking platform for the evaluation of small genomic variant callsets in terms of recall, precision, and false positive/negative error patterns. NCBench is implemented as a continuously re-evaluated open-source repository. We show that it is possible to entirely rely on public free infrastructure (Github, Github Actions, Zenodo) in combination with established open-source tools. NCBench is agnostic of the used dataset and can evaluate an arbitrary number of given callsets, while reporting the results in a visual and interactive way. We used NCBench to evaluate over 40 callsets generated by various variant calling pipelines available in the participating groups that were run on three exome datasets from different enrichment kits and at different coverages. While all pipelines achieve high overall quality, subtle systematic differences between callers and datasets exist and are made apparent by NCBench.These insights are useful to improve existing pipelines and develop new workflows. NCBench is meant to be open for the contribution of any given callset. Most importantly, for authors, it will enable the omission of repeated re-implementation of paper-specific variant calling benchmarks for the publication of new tools or pipelines, while readers will benefit from being able to (continuously) observe the performance of tools and pipelines at the time of reading instead of at the time of writing.

An overview of the goals and achievements of the European PartEmis project (Measurement and prediction of emissions of aerosols and gaseous precursors from gas turbine engines) is presented. PartEmis was focussed on the characterisation and quantification of exhaust emissions from a gas turbine engine. The engine was composed of a combustor and a unit to simulate a 3-shaft turbine section (so-called Hot End Simulator; HES). A comprehensive suite of aerosol, gas and chemi-ion measurements were conducted under different, i) combustor and HES operating conditions, ii) fuel sulphur concentrations. Measured aerosol properties were mass and number concentration, size distribution, mixing state, thermal stability of internally mixed particles, hygroscopicity, cloud condensation nuclei (CCN) activation potential, and chemical composition. Furthermore, chemi-ions, non-methane volatile organic compounds (NMVOCs) and OH were monitored. The combustor operation conditions corresponded to modern and older engine gas path temperatures at cruise altitude, with fuel sulphur contents (FSC) of 0.05, 0.41, and 1.270 g kg−1. The combustor behaved like a typical aircraft engine combustor with respect to thermodynamic data and main emissions, which suggests that the PartEmis database may be applicable to contemporary aircraft engines. The conclusions drawn from the PartEmis experiment are discussed separately for combustion particles, ultra fine particles, sulphate-containing species and chemi-ions, particle hygrioscopic growth and CCN activation, gaseous organic fraction, and emission properties.

BACKGROUND: The purpose of the study is to elucidate the sequence composition of the short arm of rye chromosome 1 (Secale cereale) with special focus on its gene content, because this portion of the rye genome is an integrated part of several hundreds of bread wheat varieties worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Multiple Displacement Amplification of 1RS DNA, obtained from flow sorted 1RS chromosomes, using 1RS ditelosomic wheat-rye addition line, and subsequent Roche 454FLX sequencing of this DNA yielded 195,313,589 bp sequence information. This quantity of sequence information resulted in 0.43× sequence coverage of the 1RS chromosome arm, permitting the identification of genes with estimated probability of 95%. A detailed analysis revealed that more than 5% of the 1RS sequence consisted of gene space, identifying at least 3,121 gene loci representing 1,882 different gene functions. Repetitive elements comprised about 72% of the 1RS sequence, Gypsy/Sabrina (13.3%) being the most abundant. More than four thousand simple sequence repeat (SSR) sites mostly located in gene related sequence reads were identified for possible marker development. The existence of chloroplast insertions in 1RS has been verified by identifying chimeric chloroplast-genomic sequence reads. Synteny analysis of 1RS to the full genomes of Oryza sativa and Brachypodium distachyon revealed that about half of the genes of 1RS correspond to the distal end of the short arm of rice chromosome 5 and the proximal region of the long arm of Brachypodium distachyon chromosome 2. Comparison of the gene content of 1RS to 1HS barley chromosome arm revealed high conservation of genes related to chromosome 5 of rice. CONCLUSIONS: The present study revealed the gene content and potential gene functions on this chromosome arm and demonstrated numerous sequence elements like SSRs and gene-related sequences, which can be utilised for future research as well as in breeding of wheat and rye.

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.

We present the results of the human genomic small variant calling benchmarking initiative of the German Research Foundation (DFG) funded Next Generation Sequencing Competence Network (NGS-CN) and the German Human Genome-Phenome Archive (GHGA). In this effort, we developed NCBench, a continuous benchmarking platform for the evaluation of small genomic variant callsets in terms of recall, precision, and false positive/negative error patterns. NCBench is implemented as a continuously re-evaluated open-source repository. We show that it is possible to entirely rely on public free infrastructure (Github, Github Actions, Zenodo) in combination with established open-source tools. NCBench is agnostic of the used dataset and can evaluate an arbitrary number of given callsets, while reporting the results in a visual and interactive way. We used NCBench to evaluate over 40 callsets generated by various variant calling pipelines available in the participating groups that were run on three exome datasets from different enrichment kits and at different coverages. While all pipelines achieve high overall quality, subtle systematic differences between callers and datasets exist and are made apparent by NCBench.These insights are useful to improve existing pipelines and develop new workflows. NCBench is meant to be open for the contribution of any given callset. Most importantly, for authors, it will enable the omission of repeated re-implementation of paper-specific variant calling benchmarks for the publication of new tools or pipelines, while readers will benefit from being able to (continuously) observe the performance of tools and pipelines at the time of reading instead of at the time of writing.