kClust: fast and sensitive clustering of large protein sequence databases

Background: Fueled by rapid progress in high-throughput sequencing, the size of public sequence databases doubles every two years. Searching the ever larger and more redundant databases is getting increasingly inefficient. Clustering can help to organize sequences into homologous and functionally similar groups and can improve the speed, sensitivity, and readability of homology searches. However, because the clustering time is quadratic in the number of sequences, standard sequence search methods are becoming impracticable. Results: Here we present a method to cluster large protein sequence databases such as UniProt within days down to 20\%-30\% maximum pairwise sequence identity. kClust owes its speed and sensitivity to an alignment-free prefilter that calculates the cumulative score of all similar 6-mers between pairs of sequences, and to a dynamic programming algorithm that operates on pairs of similar 4-mers. To increase sensitivity further, kClust can run in profile-sequence comparison mode, with profiles computed from the clusters of a previous kClust iteration. kClust is two to three orders of magnitude faster than clustering based on NCBI BLAST, and on multidomain sequences of 20\%-30\% maximum pairwise sequence identity it achieves comparable sensitivity and a lower false discovery rate. It also compares favorably to CD-HIT and UCLUST in terms of false discovery rate, sensitivity, and speed. Conclusions: kClust fills the need for a fast, sensitive, and accurate tool to cluster large protein sequence databases to below 30\% sequence identity. kClust is freely available under GPL at ftp://toolkit.lmb.uni-muenchen.de/pub/kClust/

An Improved Estimate of the Coupled Arctic Energy Budget

This study combines state-of-the-art reanalyses such as the fifth-generation European Re-Analysis (ERA5) and the Ocean Reanalysis System 5 (ORAS5) with novel observational products to present an updated estimate of the coupled atmosphere–ocean–sea ice Arctic energy budget, including flux and storage terms covering 2001–17. Observational products provide independent estimates of crucial budget terms, including oceanic heat transport from unique mooring-derived data, radiative fluxes from satellites, and sea ice volume from merged satellite data. Results show that the time averages of independent estimates of radiative, atmospheric, and oceanic energy fluxes into the Arctic Ocean domain are remarkably consistent in the sense that their sum closely matches the observed rate of regional long-term oceanic heat accumulation of ~1 W m−2. Atmospheric and oceanic heat transports are found to be stronger compared to earlier assessments (~100 and ~16 W m−2, respectively). Data inconsistencies are larger when considering the mean annual cycle of the coupled energy budget, with RMS values of the monthly budget residual between 7 and 15 W m−2, depending on the employed datasets. This nevertheless represents an average reduction of ~72% of the residual compared to earlier work and demonstrates the progress made in data quality and diagnostic techniques. Finally, the budget residual is eliminated using a variational approach to provide a best estimate of the mean annual cycle. The largest remaining sources of uncertainty are ocean heat content and latent heat associated with sea ice melt and freeze, which both suffer from the lack of observational constraints. More ocean in situ observations and reliable sea ice thickness observations and their routinely assimilation into reanalyses are needed to further reduce uncertainty.publishedVersio

3.9 Semi-field testing of the solitary bee Osmia bicornis (L., 1758) (Hymenoptera, Megachilidae) in flowering Phacelia tanacetifolia – Chances, improvements and limitations

Based on the proposed test design of the ICPPR non-Apis working group, a semi-field (tunnel) study was conducted with the solitary bee Osmia bicornis (L., 1758) in flowering Phacelia using tunnels. Untreated crop was used as a control, and the insect growth regulator fenoxycarb as a test item which is intended to be used as reference item for this study type. To improve the design and to enhance the informative value of such studies our method deviated in three points: placing the cocoons in the tubes of the nesting units, performing additional brood assessments in two to three day intervals and increasing the application rate of the test item.Overall the results indicated that the proposed test design is suitable to perform studies on O. bicornis in Phacelia under semi-field conditions.Data on the reproduction performance, brood termination and hatching rate of the progenies show low variability between the replicates in both treatment groups. No impact on the flight activity, mortality, reproduction performance and hatching success of the progenies was observed, but an increased brood termination rate of larvae produced within the first days after application was recorded; in particular, placing the cocoons in the tubes lead to higher proportions of nesting established females. Moreover, due to the assessment of the cell production in 2 to 3 day intervals, it is possible to analyse time dependent effects on the reproductive performance, brood termination and hatching rate which can be expected by the decreasing exposure in the course of the study. And finally, it is shown that fenoxycarb is not a suitable reference item for such studies.Based on the proposed test design of the ICPPR non-Apis working group, a semi-field (tunnel) study was conducted with the solitary bee Osmia bicornis (L., 1758) in flowering Phacelia using tunnels. Untreated crop was used as a control, and the insect growth regulator fenoxycarb as a test item which is intended to be used as reference item for this study type. To improve the design and to enhance the informative value of such studies our method deviated in three points: placing the cocoons in the tubes of the nesting units, performing additional brood assessments in two to three day intervals and increasing the application rate of the test item.Overall the results indicated that the proposed test design is suitable to perform studies on O. bicornis in Phacelia under semi-field conditions.Data on the reproduction performance, brood termination and hatching rate of the progenies show low variability between the replicates in both treatment groups. No impact on the flight activity, mortality, reproduction performance and hatching success of the progenies was observed, but an increased brood termination rate of larvae produced within the first days after application was recorded; in particular, placing the cocoons in the tubes lead to higher proportions of nesting established females. Moreover, due to the assessment of the cell production in 2 to 3 day intervals, it is possible to analyse time dependent effects on the reproductive performance, brood termination and hatching rate which can be expected by the decreasing exposure in the course of the study. And finally, it is shown that fenoxycarb is not a suitable reference item for such studies

Species- and sex-dependent changes in body size between 1892 and 2017, and recent biochemical signatures in rural and urban populations of two ground beetle species

Increasing urbanisation and intensified agriculture lead to rapid transitions of ecosystems. Species that persist throughout rapid transitions may respond to environmental changes across space and/or time, for instance by altering morphological and/or biochemical traits. We used natural history museum specimens, covering the Anthropocene epoch, to obtain long-term data combined with recent samples. We tested whether rural and urban populations of two ground beetle species, Harpalus affinis and H. rufipes, exhibit spatio-temporal intraspecific differences in body size. On a spatial scale, we tested signatures of nitrogen and carbon stable isotopes enrichments in different tissues and body components in recent populations of both species from urban and agricultural habitats. For body size examinations, we used beetles, collected from the early 20th century until 2017 in the Berlin-Brandenburg region, Germany, where urbanisation and agriculture have intensified throughout the last century. For stable isotope examinations, we used recent beetles from urban and agricultural habitats. Our results revealed no spatio-temporal changes in body size in both species' females. Body size of H. rufipes males decreased in the city but remained constant in rural areas over time. We discuss our findings with respect to habitat quality, urban heat and interspecific differences in activity pattern. Although nitrogen isotope ratios were mostly higher in specimens from agricultural habitats, some urban beetles reached equal enrichments. Carbon signatures of both species did not differ between habitats, detecting no differences in energy sources. Our results indicate that increasing urbanisation and intensified agriculture are influencing species' morphology and/or biochemistry. However, changes may be species- and sex-specific

Induction of T helper 2 cell responses against Schistosoma mansoni eggs in the murine intestine

T helper 2 (Th2) cell responses typify the immune response to parasitic organisms, which frequently invade the intestine. Dendritic cells (DCs) are considered vital for the induction of Th2 responses as they present parasite- derived antigens to naive T cells in draining lymph nodes. However, the identities of the DC populations responsible for priming Th2 cells in the intestine are still unclear. We developed an experimental immunization protocol to deliver Schistosoma mansoni eggs into the intestine. During live infection by the parasite, these eggs cause intestinal damage, granuloma formation, tissue fibrosis and strong type 2 immune responses. Many aspects of type 2 immunity are controlled by the transcription factor IRF4 and we observed that intestinal Th2 responses against Schistosoma mansoni eggs did not develop in the draining lymph nodes in the absence of IRF4+ DCs. IRF4f/f CD11c-cre positive mice had fewer CD11b-expessing migrating DCs, and fewer parasite antigen-carrying DCs were present in the mesenteric lymph nodes (MLNs) draining the small intestine and colon. However, transfer of antigen-loaded IRF4-deficient DCs directly into the MLN revealed that these cells could induce antigen-specific Th2 responses, suggesting that IRF4 controlled the migration of CD11b-expessing DCs rather than their Th2 inducing capacity. Furthermore, immature DCs from the intestinal lamina propria, and semi-mature DCs from lymph were sufficient to prime antigen-specific Th2 responses against egg antigens when transferred into naive recipient mice. This induction was dependent on MHCII expression but not on the production of IL-4 by the transferred DCs, indicating that conventional intestinal DCs are fully capable of inducing Th2 responses against S. mansoni egg antigens upon transfer. Further analysis of migratory small intestinal and colonic lymph DCs revealed that distinct subsets of CD11b-expressing DCs were sufficient for the induction of Th2 responses in the small intestine and colon. CD11b+CD103+ DCs transported parasite antigen from the small intestine, whereas CD11b+CD103- DCs performed this role in the colon. Of note, these same small intestinal and colonic DC subsets were also the populations that were most efficient at priming antigen-specific Th2 responses in vivo. Thus, we have not only identified that IRF4-dependent CD11b-expressing DCs are specialized to drive Th2 responses in the intestine but have also revealed that different DC subsets promote Th2 responses in the small intestine and colon. These findings not only advance our knowledge of intestinal Th2 responses against parasite antigens but also reveal a hitherto unappreciated functional heterogeneity among intestinal DCs, which could also be relevant for other tissue- specific intestinal conditions like Crohn’s disease, ulcerative colitis and celiac disease

Metastases of cutaneous squamous cell carcinoma seem to be the most frequent malignancies in the parotid gland: a hospital-based study from a salivary gland center

Malignant parotid tumors account for approximately 20% of all parotid lesions. In addition to the various primary parotid lesions there are secondary parotid malignancies, such as metastases or lymphomas. Data on histopathological distribution of all malignancies—including secondary parotid lesions—is limited. Recent evidence indicated a rising surgical incidence of secondary parotid malignancies. This study aims to review the distribution of malignancies in parotid resections from a salivary gland center. A retrospective review of prospectively collected data for all patients who had received parotidectomy between 2014 and 2019 was performed. Histopathological distribution was displayed separately for all parotid malignancies and for primary parotid malignancies. Further, patients` characteristics were compared between benign and malignant parotid lesions and between the two most common malignant parotid lesions. Out of 777 patients, 614 (78.9%) patients had a benign and 164 (21.1%) patients had a malignant parotid lesion. The most common parotid malignancy was metastatic cutaneous squamous cell carcinoma (cSCC) accounting for 35.4% of all parotid malignancies. 71.5% of all malignant lesions were secondary malignancies. Patients with metastatic cSCC were significantly older (p < 0.001) and significantly more likely to be male (p < 0.001) than patients with primary parotid malignancies. No significant difference was found when the lesion size of metastatic cSCC was compared to primary parotid malignancies (p = 0.216). The present study shows the high prevalence of secondary parotid malignancies in patients who had received parotidectomy. Furthermore, it confirms a rising surgical incidence of metastatic cSCC to the parotid gland in a series from a salivary gland center. At this time, parotid surgery for malignant lesions is more likely to be performed for metastases than for primary parotid malignancies