Fipronil Affects Craniofacial and Heart Development in Zebrafish Embryos (Danio rerio).

Fipronil is a broad-spectrum insecticide that has off-target effects in developing vertebrate embryos. In this study, we investigate treatment of zebrafish embryos with fipronil over the course of 5 days and examine the effects on body length, the cardiovascular system, and craniofacial morphology. We found the insecticide caused shorter body length and a decrease in eye size. By examining specific heart chamber morphology, as well as jaw angle and length, we quantified defects including enlargement of the heart and increases in jaw length and width. Further studies are needed to assess the mechanisms of fipronil's effect on vertebrate development for both environmental and human health concerns.

Fcgr2b and Fcgr3 are the major genetic factors for cartilage antibody-induced arthritis, overriding the effect of Hc encoding complement C5.

Like rheumatoid arthritis (RA) in humans, collagen-induced arthritis (CIA) in mice is associated with not only MHC class II genetic polymorphism but also, to some extent, with other loci including genes encoding Fc gamma receptors (FCGRs) and complement C5. In this study, we used a cartilage antibody-induced arthritis (CAIA) model in which arthritis develops within a 12-h timeframe, to determine the relative importance of FCGRs and C5 (Hc). In CAIA, inhibiting or deleting FCGR3 substantially hindered arthritis development, underscoring the crucial role of this receptor. Blocking FCGR3 also reduced the levels of FCGR4, and vice versa. When employing an IgG1 arthritogenic cocktail that exclusively interacts with FCGR2B and FCGR3, joint inflammation was promptly initiated in Fcgr2b-- mice but not in Fcgr3-- mice, suggesting that FCGR3 is sufficient for CAIA development. Regarding complement activation, Fcgr2b++.Hc** mice with C5 mutated were fully resistant to CAIA, whereas Fcgr2b--.Hc** mice developed arthritis rapidly. We conclude that FCGR3 is essential and sufficient for CAIA development, particularly when induced by IgG1 antibodies. The human ortholog of mouse FCGR3, FCGR2A, may be associated with RA pathogenesis. FCGR2B deficiency allows for rapid arthritis progression and overrides the resistance conferred by C5 deficiency.

A subset of type-II collagen-binding antibodies prevents experimental arthritis by inhibiting FCGR3 signaling in neutrophils.

Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1ß secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA.

Antigen-presenting autoreactive B cells activate regulatory T cells and suppress autoimmune arthritis in mice.

B cells undergo several rounds of selection to eliminate potentially pathogenic autoreactive clones, but in contrast to T cells, evidence of positive selection of autoreactive B cells remains moot. Using unique tetramers, we traced natural autoreactive B cells (C1-B) specific for a defined triple-helical epitope on collagen type-II (COL2), constituting a sizeable fraction of the physiological B cell repertoire in mice, rats, and humans. Adoptive transfer of C1-B suppressed arthritis independently of IL10, separating them from IL10-secreting regulatory B cells. Single-cell sequencing revealed an antigen processing and presentation signature, including induced expression of CD72 and CCR7 as surface markers. C1-B presented COL2 to T cells and induced the expansion of regulatory T cells in a contact-dependent manner. CD72 blockade impeded this effect suggesting a new downstream suppressor mechanism that regulates antigen-specific T cell tolerization. Thus, our results indicate that autoreactive antigen-specific naïve B cells tolerize infiltrating T cells against self-antigens to impede the development of tissue-specific autoimmune inflammation.

Online Phylogenetics with matOptimize Produces Equivalent Trees and is Dramatically More Efficient for Large SARS-CoV-2 Phylogenies than de novo and Maximum-Likelihood Implementations.

Phylogenetics has been foundational to SARS-CoV-2 research and public health policy, assisting in genomic surveillance, contact tracing, and assessing emergence and spread of new variants. However, phylogenetic analyses of SARS-CoV-2 have often relied on tools designed for de novo phylogenetic inference, in which all data are collected before any analysis is performed and the phylogeny is inferred once from scratch. SARS-CoV-2 data sets do not fit this mold. There are currently over 14 million sequenced SARS-CoV-2 genomes in online databases, with tens of thousands of new genomes added every day. Continuous data collection, combined with the public health relevance of SARS-CoV-2, invites an "online" approach to phylogenetics, in which new samples are added to existing phylogenetic trees every day. The extremely dense sampling of SARS-CoV-2 genomes also invites a comparison between likelihood and parsimony approaches to phylogenetic inference. Maximum likelihood (ML) and pseudo-ML methods may be more accurate when there are multiple changes at a single site on a single branch, but this accuracy comes at a large computational cost, and the dense sampling of SARS-CoV-2 genomes means that these instances will be extremely rare because each internal branch is expected to be extremely short. Therefore, it may be that approaches based on maximum parsimony (MP) are sufficiently accurate for reconstructing phylogenies of SARS-CoV-2, and their simplicity means that they can be applied to much larger data sets. Here, we evaluate the performance of de novo and online phylogenetic approaches, as well as ML, pseudo-ML, and MP frameworks for inferring large and dense SARS-CoV-2 phylogenies. Overall, we find that online phylogenetics produces similar phylogenetic trees to de novo analyses for SARS-CoV-2, and that MP optimization with UShER and matOptimize produces equivalent SARS-CoV-2 phylogenies to some of the most popular ML and pseudo-ML inference tools. MP optimization with UShER and matOptimize is thousands of times faster than presently available implementations of ML and online phylogenetics is faster than de novo inference. Our results therefore suggest that parsimony-based methods like UShER and matOptimize represent an accurate and more practical alternative to established ML implementations for large SARS-CoV-2 phylogenies and could be successfully applied to other similar data sets with particularly dense sampling and short branch lengths.

Pathogenic antibody response to glucose-6-phosphate isomerase targets a modified epitope uniquely exposed on joint cartilage.

OBJECTIVES: To identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA). METHODS: IgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining. RESULTS: Peptide GPI293-307 was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI293-307 epitopes, and high affinity anti-GPI293-307 IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI293-307 IgG antibodies induced arthritis in mice. Moreover, anti-GPI293-307 IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI293-307-specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab-peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI293-307 antibodies. CONCLUSIONS: We have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293-307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA.

Treenome Browser: co-visualization of enormous phylogenies and millions of genomes.

SUMMARY: Treenome Browser is a web browser tool to interactively visualize millions of genomes alongside huge phylogenetic trees. AVAILABILITY AND IMPLEMENTATION: Treenome Browser for SARS-CoV-2 can be accessed at cov2tree.org, or at taxonium.org for user-provided trees. Source code and documentation are available at github.com/theosanderson/taxonium and docs.taxonium.org/en/latest/treenome.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transposable elements drive intron gain in diverse eukaryotes.

There is massive variation in intron numbers across eukaryotic genomes, yet the major drivers of intron content during evolution remain elusive. Rapid intron loss and gain in some lineages contrast with long-term evolutionary stasis in others. Episodic intron gain could be explained by recently discovered specialized transposons called Introners, but so far Introners are only known from a handful of species. Here, we performed a systematic search across 3,325 eukaryotic genomes and identified 27,563 Introner-derived introns in 175 genomes (5.2%). Species with Introners span remarkable phylogenetic diversity, from animals to basal protists, representing lineages whose last common ancestor dates to over 1.7 billion years ago. Aquatic organisms were 6.5 times more likely to contain Introners than terrestrial organisms. Introners exhibit mechanistic diversity but most are consistent with DNA transposition, indicating that Introners have evolved convergently hundreds of times from nonautonomous transposable elements. Transposable elements and aquatic taxa are associated with high rates of horizontal gene transfer, suggesting that this combination of factors may explain the punctuated and biased diversity of species containing Introners. More generally, our data suggest that Introners may explain the episodic nature of intron gain across the eukaryotic tree of life. These results illuminate the major source of ongoing intron creation in eukaryotic genomes.

NCF4 dependent intracellular reactive oxygen species regulate plasma cell formation.

Defective reactive oxygen species (ROS) production by genetically determined variants of the NADPH oxidase 2 (NOX2) complex component, NCF4, leads to enhanced production of autoantibodies to collagen type II (COL2) and severe collagen-induced arthritis (CIA) in mice. To further understand this process, we used mice harboring a mutation in the lipid endosomal membrane binding site (R58A) of NCF4 subunit. This mutation did not affect the extracellular ROS responses but showed instead decreased intracellular responses following B cell stimulation. Immunization with COL2 led to severe arthritis with increased antibody levels in Ncf458A mutated animals without significant effects on antigen presentation, autoreactive T cell activation and germinal center formation. Instead, plasma cell formation was enhanced and had altered CXCR3/CXCR4 expression. This B cell intrinsic effect was further confirmed with chimeric B cell transfer experiments and in vitro LPS or CD40L with anti-IgM stimulation. We conclude that NCF4 regulates the terminal differentiation of B cells to plasma cells through intracellular ROS.

matOptimize: a parallel tree optimization method enables online phylogenetics for SARS-CoV-2.

MOTIVATION: Phylogenetic tree optimization is necessary for precise analysis of evolutionary and transmission dynamics, but existing tools are inadequate for handling the scale and pace of data produced during the coronavirus disease 2019 (COVID-19) pandemic. One transformative approach, online phylogenetics, aims to incrementally add samples to an ever-growing phylogeny, but there are no previously existing approaches that can efficiently optimize this vast phylogeny under the time constraints of the pandemic. RESULTS: Here, we present matOptimize, a fast and memory-efficient phylogenetic tree optimization tool based on parsimony that can be parallelized across multiple CPU threads and nodes, and provides orders of magnitude improvement in runtime and peak memory usage compared to existing state-of-the-art methods. We have developed this method particularly to address the pressing need during the COVID-19 pandemic for daily maintenance and optimization of a comprehensive SARS-CoV-2 phylogeny. matOptimize is currently helping refine on a daily basis possibly the largest-ever phylogenetic tree, containing millions of SARS-CoV-2 sequences. AVAILABILITY AND IMPLEMENTATION: The matOptimize code is freely available as part of the UShER package (https://github.com/yatisht/usher) and can also be installed via bioconda (https://bioconda.github.io/recipes/usher/README.html). All scripts we used to perform the experiments in this manuscript are available at https://github.com/yceh/matOptimize-experiments. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Online Phylogenetics using Parsimony Produces Slightly Better Trees and is Dramatically More Efficient for Large SARS-CoV-2 Phylogenies than de novo and Maximum-Likelihood Approaches.

Phylogenetics has been foundational to SARS-CoV-2 research and public health policy, assisting in genomic surveillance, contact tracing, and assessing emergence and spread of new variants. However, phylogenetic analyses of SARS-CoV-2 have often relied on tools designed for de novo phylogenetic inference, in which all data are collected before any analysis is performed and the phylogeny is inferred once from scratch. SARS-CoV-2 datasets do not fit this mould. There are currently over 10 million sequenced SARS-CoV-2 genomes in online databases, with tens of thousands of new genomes added every day. Continuous data collection, combined with the public health relevance of SARS-CoV-2, invites an "online" approach to phylogenetics, in which new samples are added to existing phylogenetic trees every day. The extremely dense sampling of SARS-CoV-2 genomes also invites a comparison between likelihood and parsimony approaches to phylogenetic inference. Maximum likelihood (ML) methods are more accurate when there are multiple changes at a single site on a single branch, but this accuracy comes at a large computational cost, and the dense sampling of SARS-CoV-2 genomes means that these instances will be extremely rare because each internal branch is expected to be extremely short. Therefore, it may be that approaches based on maximum parsimony (MP) are sufficiently accurate for reconstructing phylogenies of SARS-CoV-2, and their simplicity means that they can be applied to much larger datasets. Here, we evaluate the performance of de novo and online phylogenetic approaches, and ML and MP frameworks, for inferring large and dense SARS-CoV-2 phylogenies. Overall, we find that online phylogenetics produces similar phylogenetic trees to de novo analyses for SARS-CoV-2, and that MP optimizations produce more accurate SARS-CoV-2 phylogenies than do ML optimizations. Since MP is thousands of times faster than presently available implementations of ML and online phylogenetics is faster than de novo , we therefore propose that, in the context of comprehensive genomic epidemiology of SARS-CoV-2, MP online phylogenetics approaches should be favored.

Pandemic-scale phylogenetics.

Phylogenetics has been central to the genomic surveillance, epidemiology and contact tracing efforts during the COVD-19 pandemic. But the massive scale of genomic sequencing has rendered the pre-pandemic tools inadequate for comprehensive phylogenetic analyses. Here, we discuss the phylogenetic package that we developed to address the needs imposed by this pandemic. The package incorporates several pandemic-specific optimization and parallelization techniques and comprises four programs: UShER, matOptimize, RIPPLES and matUtils. Using high-performance computing, UShER and matOptimize maintain and refine daily a massive mutation-annotated phylogenetic tree consisting of all SARS-CoV-2 sequences available in online repositories. With UShER and RIPPLES, individual labs - even with modest compute resources - incorporate newly-sequenced SARS-CoV-2 genomes on this phylogeny and discover evidence for recombination in real-time. With matUtils, they rapidly query and visualize massive SARS-CoV-2 phylogenies. These tools have empowered scientists worldwide to study the SARS-CoV-2 evolution and transmission at an unprecedented scale, resolution and speed.

Influence of Process Parameters on Grain Size and Texture Evolution of Fe-3.2 wt.-% Si Non-Oriented Electrical Steels.

The magnetic properties of non-oriented electrical steel, widely used in electric machines, are closely related to the grain size and texture of the material. How to control the evolution of grain size and texture through processing in order to improve the magnetic properties is the research focus of this article. Therefore, the complete process chain of a non-oriented electrical steel with 3.2 wt.-% Si was studied with regard to hot rolling, cold rolling, and final annealing on laboratory scale. Through a comprehensive analysis of the process chain, the influence of important process parameters on the grain size and texture evolution as well as the magnetic properties was determined. It was found that furnace cooling after the last hot rolling pass led to a fully recrystallized grain structure with the favorable ND-rotated-cube component, and a large portion of this component was retained in the thin strip after cold rolling, resulting in a texture with a low γ-fiber and a high ND-cube component after final annealing at moderate to high temperatures. These promising results on a laboratory scale can be regarded as an effective way to control the processing on an industrial scale, to finally tailor the magnetic properties of non-oriented electrical steel according to their final application.

A Daily-Updated Database and Tools for Comprehensive SARS-CoV-2 Mutation-Annotated Trees.

The vast scale of SARS-CoV-2 sequencing data has made it increasingly challenging to comprehensively analyze all available data using existing tools and file formats. To address this, we present a database of SARS-CoV-2 phylogenetic trees inferred with unrestricted public sequences, which we update daily to incorporate new sequences. Our database uses the recently proposed mutation-annotated tree (MAT) format to efficiently encode the tree with branches labeled with parsimony-inferred mutations, as well as Nextstrain clade and Pango lineage labels at clade roots. As of June 9, 2021, our SARS-CoV-2 MAT consists of 834,521 sequences and provides a comprehensive view of the virus' evolutionary history using public data. We also present matUtils-a command-line utility for rapidly querying, interpreting, and manipulating the MATs. Our daily-updated SARS-CoV-2 MAT database and matUtils software are available at http://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/UShER_SARS-CoV-2/ and https://github.com/yatisht/usher, respectively.

Vegetation traits of pre-Alpine grasslands in southern Germany.

The data set contains information on aboveground vegetation traits of > 100 georeferenced locations within ten temperate pre-Alpine grassland plots in southern Germany. The grasslands were sampled in April 2018 for the following traits: bulk canopy height; weight of fresh and dry biomass; dry weight percentage of the plant functional types (PFT) non-green vegetation, legumes, non-leguminous forbs, and graminoids; total green area index (GAI) and PFT-specific GAI; plant water content; plant carbon and nitrogen content (community values and PFT-specific values); as well as leaf mass per area (LMA) of PFT. In addition, a species specific inventory of the plots was conducted in June 2020 and provides plot-level information on grassland type and plant species composition. The data set was obtained within the framework of the SUSALPS project ("Sustainable use of alpine and pre-alpine grassland soils in a changing climate"; https://www.susalps.de/ ) to provide in-situ data for the calibration and validation of remote sensing based models to estimate grassland traits.

Specific Varicella-Related Complications and Their Decrease in Hospitalized Children after the Introduction of General Varicella Vaccination: Results from a Multicenter Pediatric Hospital Surveillance Study in Bavaria (Germany).

BACKGROUND: Universal varicella vaccination (UVV) for children introduced in Germany in 2004 resulted in a significant overall decline of varicella-related hospitalizations (VRHs). We investigated the incidence of specific types of varicella-related complications (VRCs) in hospitalized children and the impact of UVV on VRCs during the first 7 years of UVV. METHODS: Children < 17 years of age hospitalized with an ICD-10-based (International Classification of Diseases, 10th Revision) discharge diagnosis of varicella were identified as VRH in pediatric hospitals in Bavaria by annual standardized data queries of the hospital databases (2005-2011). For each VRH, the hospitals reported basic demographic data, duration of hospital stay, all diagnostic and procedural codes, and outcome. VRCs were reported overall, per year, and by immune status. Complication rates were calculated as mean number per complication category per hospital and per year; VRC trends over time were assessed by linear regression. RESULTS: Between 78% (2005) and 61% (2011) of Bavarian hospitals participated and reported a total of 1263 VRHs. Specific VRCs were reported in 954 (76%) children. Complication rates per hospital and year decreased from 6.7 [95% confidence interval (CI): 5.1-8.3] in 2005 to 1.5 (95% CI: 0.8-2.3) in 2011, with the strongest reduction of 90% in children < 5 years of age from 5.3 (95% CI: 4.0-6.6) in 2005 to 0.5 (95% CI: 0.1-0.9) in 2011. Significant decreases were observed for children with upper respiratory tract (URT, by 97%), lower respiratory tract (LRT, by 90%), skin (by 81%), gastrointestinal (by 78%), and neurologic (by 65%) VRCs. Forty-eight children with VRCs were immunocompromised; their annual rate decreased by 87%. DISCUSSION: Corresponding to increasing varicella vaccination coverage in the population, the incidence of VRC decreased by 77% from 2005 to 2011, with the most substantial decrease in the target group for UVV. CONCLUSION: Within 7 years, UVV in Germany led to a decrease of about 77% of all types of VRCs, with the highest reductions observed for VRCs of the respiratory tract.

[Assessment of health risks due to heat and fine particles in Germany: an epidemiological study approach]. / Erfassung von hitze- und feinstaubbedingten Gesundheitsrisiken in Deutschland: Ein epidemiologischer Studienansatz.

BACKGROUND: Exposure to heat and particulate matter is a cause of increased mortality. Climate change and increasing climate variability exacerbate these problems. Experts require assessments with which health risks and the success of preventative measures can be estimated. We implemented an ecological study approach to assess these risks at both small and large scales of reference levels (Federal Republic of Germany and territorial authority). METHODS: We utilised a case-crossover design to investigate the relationship between exposure and mortality. This study design uses a logistic regression model. Analogously to a matched case-control study, the odds ratio maps the effect strength. The study period included the years 2002-2006. RESULTS: The analysis demonstrated health risks from exposure to heat for the German population (OR 1.1529, 95% CI 1.1517-1.1541; adjusted OR 1.0658). Significant evidence of a health risk was also documented for exposure to particulate matter (PM10; OR 1.2987, 95% CI 1.2951-1.3024; adjusted OR 1.0128). The risk does not significantly differ for women versus men; the variable age was also not significant at the level of the country-wide analysis, but for a few subordinate units of space. This study approach can be adapted for assessments at varying levels of reference and periods of time as well as for different populations. DISCUSSION: The methodological approach is useful for a reproducible study design. Nevertheless, other influencing factors such as ozone or PM2.5 should be incorporated in subsequent analyses to clarify whether these factors skew the results. Further analysis would also be useful to investigate if and to what extent socio-structural and socio-economic factors affect the associated risk.

[INTRAOPERATIVE RADIOTHERAPY (IORT) IN EARLY BREAST CANCER - 500 PATIENTS, ONE CENTER'S EXPERIENCE].

AIMS: To present our Institute's experience with intraoperative radiotherapy in this selected population by collecting and analyzing clinical data, including long-term follow-up. BACKGROUND: Breast-conserving therapy is the standard treatment for early-stage breast cancer. The treatment includes tumor resection and a whole breast irradiation. Intraoperative radiotherapy is a single dose of irradiation given to the tumor bed immediately after it is removed. This treatment is suitable for a selected population of patients with early stage breast cancer, which constitutes about 20% of all breast cancer patients and is supposed to replace the standard whole breast radiation treatment. METHODS: Between the years 2006-2017, 737 women with early breast cancer were treated in Carmel Medical Center with intraoperative radiotherapy. We herein report the results of the first 500 patients who were treated until 2015. RESULTS: In 13.8% of the patients, additional breast treatment was recommended due to poor pathological characteristics of the disease in final pathological examination. During a median follow-up period of 74 months (1-136), recurrence was observed in 22 patients (4.4%), and in 7 patients (1.4%) recurrence was observed in regional lymph nodes; 13 patients (2.6%) developed metastatic disease. Risk factors for regional recurrence were identified: tumor size greater than 2 cm, lack of adjuvant therapy and poor genetic profile of the disease. CONCLUSIONS: Intraoperative radiotherapy is feasible and may offer an alternative to the standard whole breast radiotherapy, in low risk early breast cancer patients. The patients should be selected according to known risk factors.