Comparative transcriptomics illuminates the cellular responses of an aeroterrestrial zygnematophyte to UV radiation.

The zygnematophytes are the closest relatives of land plants and comprise several lineages that adapted to a life on land. Species of the genus Serritaenia form colorful, mucilaginous capsules, which surround the cells and block harmful solar radiation, one of the major terrestrial stressors. In eukaryotic algae, this "sunscreen mucilage" represents a unique photoprotective strategy, whose induction and chemical background are unknown. We generated a de novo transcriptome of Serritaenia testaceovaginata and studied its gene regulation under moderate ultraviolet radiation (UVR) that triggers sunscreen mucilage under experimental conditions. UVR induced the repair of DNA and the photosynthetic apparatus as well as the synthesis of aromatic specialized metabolites. Specifically, we observed pronounced expressional changes in the production of aromatic amino acids, phenylpropanoid biosynthesis genes, potential cross-membrane transporters of phenolics, and extracellular, oxidative enzymes. Interestingly, the most upregulated enzyme was a secreted class III peroxidase, whose embryophyte homologs are involved in apoplastic lignin formation. Overall, our findings reveal a conserved, plant-like UVR perception system (UVR8 and downstream factors) in zygnematophyte algae and point to a polyphenolic origin of Serritaenia's sunscreen pigment, whose synthesis might be extracellular and oxidative, resembling that of plant lignins.

Pseudovampyrella gen. nov.: A genus of Vampyrella-like protoplast extractors finds its place in the Leptophryidae.

Vampyrellid amoebae are predatory protists, which consume a variety of eukaryotic prey and inhabit freshwater, marine and terrestrial ecosystems. Although they have been known for almost 150 years, much of their diversity lacks an in-depth characterization. To date, environmental sequencing data hint at several uncharacterized lineages, to which no phenotype is associated. Furthermore, there are numerous historically described species without any molecular information. This study reports on two new vampyrellid strains from moorlands, which extract the protoplasts of Closterium species (Zygnematophyceae). Our data on morphology, prey range specificity and feeding strategy reveal that the studied vampyrellids are very similar to the historically described Vampyrella closterii. However, phylogenetic analyses demonstrate that the two strains do not belong to the genus Vampyrella and, instead, form a distinct clade in the family Leptophryidae. Hence, we introduce a new genus of algivorous protoplast extractors, Pseudovampyrella gen. nov., with the species P. closterii (= V. closterii) and P. minor. Our findings indicate that the genetic diversity of morphologically described vampyrellid species might be hugely underrated.

Purple photosymbioses.

Muñoz-Gómez and Hess introduce purple photosymbioses, which involve a heterotrophic protist host and anoxygenic photosymbionts from the phylum Proteobacteria.

Effects of multiple stressors on benthic invertebrates using Water Framework Directive monitoring data.

Multiple stressors affect freshwater systems and cause a deficient ecological status according to the European Water Framework Directive (WFD). To select effective mitigation measures and improve the ecological status, knowledge on the stressor hierarchy and individual and joined effects is necessary. However, compared to common stressors like nutrient enrichment and morphological degradation, the relative importance of micropollutants such as pesticides and pharmaceuticals is largely unaddressed. We used WFD monitoring data from Saxony (Germany) to investigate the importance of 85 environmental variables (including 34 micropollutants) for 18 benthic invertebrate metrics at 108 sites. The environmental variables were assigned to five groups (natural factors, nutrient enrichment, metals, micropollutants and morphological degradation) and were ranked according to their relative importance as group and individually within and across groups using Principal Component Analyses (PCAs) and Boosted Regression Trees (BRTs). Overall, natural factors contributed the most to the total explained deviance of the models. This variable group represented not only typological differences between sampling sites but also a gradient of human impact by strongly anthropogenically influenced variables such as electric conductivity and dissolved oxygen. These large-scale effects can mask the individual importance of the other variable groups, which may act more specifically at a subset of sites. Accordingly, micropollutants were not represented by a few dominant variables but rather a diverse palette of different chemicals with similar contribution. As a group, micropollutants contributed similarly as metals, nutrient enrichment and morphological degradation. However, the importance of micropollutants might be underestimated due to limitations of the current chemical monitoring practices.

Dose-escalated re-irradiation improves outcome in locally recurrent head and neck cancer - Results of a large multicenter analysis.

To determine efficacy and prognostic parameters of definitive re-irradiation of locoregionally recurrent squamous cell carcinoma of the head and neck (HNSCC). MATERIALS AND METHODS: Patients with locoregionally recurrent or second primary HNSCC undergoing re-irradiation with modern radiotherapy technique were eligible for this multicentric retrospective analysis. Main endpoints were overall survival (OS), progression-free survival (PFS) and locoregional control (LC). Univariate analyses were performed using the Kaplan Meier Method (log-rank). For multivariable analysis, Cox regression was used. RESULTS: A total of 253 patients treated between 2009 and 2020 at 16 university hospitals in Germany were included. The median follow up was 27.4 months (range 0.5-130). The median OS and PFS were 13.2 (CI: 10.7 - 15.7) months and 7.9 (CI: 6.7 - 9.1) months, respectively, corresponding to two-year OS and PFS rates of 29 % and 19 %. Rates of locoregional progression and "in-field-failure" were 62 % and 51 % after two years. Multivariable Cox regression analysis identified good ECOG performance status and high radiation dose as independent prognostic parameters for OS. Doses above 50 Gy (EQD2) achieved longer median OS of 17.8 months (vs 11.7 months, p < 0.01) and longer PFS of 9.6 months (vs 6.8 months, p < 0.01). In addition, there was a trend for worse survival in patients with tracheostomy (multivariable, p = 0.061). Concomitant systemic therapy did not significantly impact PFS or OS. CONCLUSION: Re-irradiation of locally recurrent or second primary HNSCC is efficient, especially if doses above 50 Gy (EQD2) are delivered. ECOG performance score was the strongest prognostic parameter for OS and PFS.

Experimental evidence for enzymatic cell wall dissolution in a microbial protoplast feeder (Orciraptor agilis, Viridiraptoridae).

BACKGROUND: Several protists have evolved the ability to perforate the cell walls of algae and fungi to specifically feed on their cell contents. These phagotrophic "protoplast feeders" represent an interesting mechanistic intermediate between predators and parasites and pose a number of cell biological questions. Although their fascinating feeding behaviour has been observed for the last 150 years, it is still unknown how protoplast feeders produce the well-defined and species-specific perforations in biochemically diverse cell walls. Differential expression analyses of the algivorous flagellate Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria) suggested the involvement of a highly expressed putative glycoside hydrolase of family GH5_5. To assess the importance of this carbohydrate-active enzyme in the feeding act of Orciraptor, we recombinantly produced its catalytic domain and studied the enzymatic activity, cellular localisation and function. RESULTS: The GH5_5 catalytic domain from Orciraptor showed pronounced activity on soluble cellulose derivatives and mixed-linkage glucans, with reaction optima comparable to known GH5_5 representatives. Crystalline cellulose was not digested by the enzyme, which suggests a typical endocellulase activity. Immunocytochemistry with a polyclonal antibody raised against the GH5_5 domain revealed that the native endocellulase localises to the contact zone of Orciraptor and the algal cell wall (= perforation zone) and to intracellular granules, which were enriched during attack. Furthermore, the anti-GH5_5 antibody applied to live cells significantly reduced the feeding success of Orciraptor. The cells attacked the algae, which, however, resulted in numerous incomplete perforations. CONCLUSIONS: Our experimental data from enzymatic assays, immunocytochemistry and inhibition experiments strongly suggest a key role of the GH5_5 endocellulase in cell wall dissolution by Orciraptor agilis. With that, we provide evidence that the well-defined perforations produced by protoplast feeders are caused by extracellular carbohydrate-active enzymes and made a first step towards establishing the molecular basis of a fascinating, yet poorly understood microbial feeding strategy.

A phylogenomically informed five-order system for the closest relatives of land plants.

The evolution of streptophytes had a profound impact on life on Earth. They brought forth those photosynthetic eukaryotes that today dominate the macroscopic flora: the land plants (Embryophyta).1 There is convincing evidence that the unicellular/filamentous Zygnematophyceae-and not the morphologically more elaborate Coleochaetophyceae or Charophyceae-are the closest algal relatives of land plants.2-6 Despite the species richness (>4,000), wide distribution, and key evolutionary position of the zygnematophytes, their internal phylogeny remains largely unresolved.7,8 There are also putative zygnematophytes with interesting body plan modifications (e.g., filamentous growth) whose phylogenetic affiliations remain unknown. Here, we studied a filamentous green alga (strain MZCH580) from an Austrian peat bog with central or parietal chloroplasts that lack discernible pyrenoids. It represents Mougeotiopsis calospora PALLA, an enigmatic alga that was described more than 120 years ago9 but never subjected to molecular analyses. We generated transcriptomic data of M. calospora strain MZCH580 and conducted comprehensive phylogenomic analyses (326 nuclear loci) for 46 taxonomically diverse zygnematophytes. Strain MZCH580 falls in a deep-branching zygnematophycean clade together with some unicellular species and thus represents a formerly unknown zygnematophycean lineage with filamentous growth. Our well-supported phylogenomic tree lets us propose a new five-order system for the Zygnematophyceae and provides evidence for at least five independent origins of true filamentous growth in the closest algal relatives of land plants. This phylogeny provides a robust and comprehensive framework for performing comparative analyses and inferring the evolution of cellular traits and body plans in the closest relatives of land plants.

Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation.

Microbial eukaryotes display a stunning diversity of feeding strategies, ranging from generalist predators to highly specialized parasites. The unicellular "protoplast feeders" represent a fascinating mechanistic intermediate, as they penetrate other eukaryotic cells (algae and fungi) like some parasites but then devour their cell contents by phagocytosis.1 Besides prey recognition and attachment, this complex behavior involves the local, pre-phagocytotic dissolution of the prey cell wall, which results in well-defined perforations of species-specific size and structure.2 Yet the molecular processes that enable protoplast feeders to overcome cell walls of diverse biochemical composition remain unknown. We used the flagellate Orciraptor agilis (Viridiraptoridae, Rhizaria) as a model protoplast feeder and applied differential gene expression analysis to examine its penetration of green algal cell walls. Besides distinct expression changes that reflect major cellular processes (e.g., locomotion and cell division), we found lytic carbohydrate-active enzymes that are highly expressed and upregulated during the attack on the alga. A putative endocellulase (family GH5_5) with a secretion signal is most prominent, and a potential key factor for cell wall dissolution. Other candidate enzymes (e.g., lytic polysaccharide monooxygenases) belong to families that are largely uncharacterized, emphasizing the potential of non-fungal microeukaryotes for enzyme exploration. Unexpectedly, we discovered various chitin-related factors that point to an unknown chitin metabolism in Orciraptor agilis, potentially also involved in the feeding process. Our findings provide first molecular insights into an important microbial feeding behavior and new directions for cell biology research on non-model eukaryotes.

German consumer evaluations of milk in blind and nonblind tests.

Greater differentiation is being made between types of milk according to product and process characteristics. This study investigated consumer preferences for 4 different types of milk in packaging-blind and nonblind experiments. The taste of long-life milk, conventional fresh whole milk, pasture-based milk, and organic milk was assessed by 138 randomly selected respondents. The average taste of packaging-blind versus nonblind milk types was statistically significantly different for each of the 4 milk types. Consumers appeared to be influenced by the milk's packaging and related credence-good attributes. It is noteworthy that the taste of organic milk during the blind tasting was given a lower rating, and the taste of long-life milk or UHT milk a higher rating, than when respondents were aware of the respective milk types. The study estimated multilevel mixed-effects logistic regression models with the response variable buying decision. Of all the factors studied in the blind and nonblind tasting of almost all milk types, buying decisions were influenced most by taste, overall assessment and income.

Farmers' preferences for adopting on-farm concentration of raw milk: Results from a discrete choice experiment in Germany.

Milk concentrates are used in the manufacturing of dairy products such as yogurt and cheese or are processed into milk powder. Processes for the nonthermal separation of water and valuable milk ingredients are becoming increasingly widespread at farm level. The technical barriers to using farm-manufactured milk concentrate in dairies are minimal, hence the suspicion that the practice of on-farm raw milk concentration is still fairly uncommon for economic reasons. This study, therefore, set out to investigate farmers' potential willingness to adopt a raw milk concentration plant. The empirical analysis was based on discrete choice experiments with 75 German dairy farmers to identify preferences and the possible adoption of on-farm raw milk concentration. The results showed that, in particular, farmers who deemed the current milk price to be insufficient viewed on-farm concentration using membrane technology as an option for diversifying their milk sales. We found no indication that adoption would be impeded by a lack of trustworthy information on milk processing technologies or capital.

The Vampyrellid Amoebae (Vampyrellida, Rhizaria).

The Vampyrellida (Endomyxa, Rhizaria) is a group of free-living, predatory amoebae, which is most closely related to the Phytomyxea (plasmodiophorids and phagomyxids). It encompasses about 50 credibly described species that have a characteristic life history with the regular alternation of trophic amoebae and immobile digestive cysts. All known vampyrellid amoebae are naked and filose, but the different species display a broad morphological variety. Vampyrellids also vary greatly in their feeding habits, and range from generalist predators to specialized 'protoplast feeders' that exclusively feed on the cell contents of eukaryotic prey. They can be found in freshwater, soil and marine habitats, and appear to be globally distributed. Yet, the phenotypic diversity and ecological roles of the Vampyrellida are still poorly explored. Currently, there are eight well-recognized subclades that comprise four families (Vampyrellidae, Leptophryidae, Placopodidae and Sericomyxidae) as well as some lineages without any phenotypic information. Research on vampyrellids is challenging due to their cryptic occurrence in nature, intricate feeding habits that complicate cultivation, and a convoluted taxonomic history. Here, we review available information about cell structure, diversity, ecology, taxonomy and phylogenetics, and provide an up-to-date introduction to the Vampyrellida that may facilitate future research.

Locomotor behavior of Neocaridina palmata: a study with leachates from UV-weathered microplastics.

Weathering of plastics leads to the formation of increasingly smaller particles with the release of chemical compounds. The latter occurs with currently unknown environmental impacts. Leachate-induced effects of weathered microplastics (MPs) are therefore of increasing concern. To investigate the toxicity of the chemical mixtures from such plastics, we exposed the freshwater shrimp Neocaridina palmata to enriched leachates from unweathered and artificially weathered (UV-A/B light) MPs (≤1 mm) from recycled low-density polyethylene (LDPE-R) pellets and from a biodegradable, not fully bio-based starch blend (SB) foil. We analyzed the individual locomotor activity (moved distance and frozen events) on day 1, 3, 7 and 14 of exposure to five leachate concentrations equivalent to 0.40-15.6 g MPs L-1, representing the upper scale of MPs that have been found in the environment. The median moved distance did not change as a function of concentration, except for the unweathered SB treatment on day 14 that indicated hyperactivity with increasing concentrations. Significant impacts were solely detected for few concentrations and exposure days. Generally, no consistent trend was observed across the experiments. We further assessed the baseline toxicity of the samples in the Microtox assay and detected high bioluminescence inhibitions of the bacterium Aliivibrio fischeri. This study demonstrates that neither the recycled nor the biodegradable material are without impacts on test parameters and therefore cannot be seen as safe alternative for conventional plastics regarding the toxicity. However, the observed in vitro toxicity did not result in substantial effects on the behavior of shrimps. Overall, we assume that the two endpoints examined in the atyid shrimp N. palmata were not sensitive to chemicals leaching from plastics or that effects on the in vivo level affect other toxic endpoints which were not considered in this study.

The amoebae of Idionectes vortex (Cutosea, Amoebozoa): Motility, cytoskeleton architecture and extracellular scales.

The Cutosea represent a deep-branching lineage within the phylum Amoebozoa that is still relatively poorly explored. Currently, there are four cutosean representatives known - the monotypic genera Armaparvus, Idionectes, Sapocribrum, and Squamamoeba - with marked genetic distances. Idionectes vortex is the deepest-branching species and differs markedly from the other Cutosea in ecology, life history, and most importantly, in its ability to form a flagellated swarmer with an exceptional swimming mechanism. As far as we know, the other Cutosea lack flagella and rather represent small, marine amoebae with a characteristic cell coat. The present paper focuses on the amoeboid life history stage of the algivorous amoeboflagellate Idionectes vortex to provide data for a first in-depth comparison with other Cutosea and to document structural specialties. The amoeboid stage of Idionectes is mainly associated with the specific feeding process, that is, the interaction with algal prey cells and phagocytosis of protoplast material. Yet, the present data from time-lapse microscopy, cytochemical stainings, and electron microscopy demonstrate clear similarities with the other cutosean species concerning amoeboid locomotion and cell coat ultrastructure. Furthermore, Idionectes amoebae exhibit a well-developed microtubular cytoskeleton, and an unusual basal apparatus that seems to undergo marked changes during the life history of this exceptional amoebozoan.

Description of the marine predator Sericomyxa perlucida gen. et sp. nov., a cultivated representative of the deepest branching lineage of vampyrellid amoebae (Vampyrellida, Rhizaria).

The vampyrellids (Vampyrellida, Rhizaria) are naked amoebae of considerable genetic diversity. Three families have been well-defined (Vampyrellidae, Leptophryidae, and Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterized. In the brackish sediment of Lake Bras D'Or, Nova Scotia, Canada, we discovered an amoeba with a vampyrellid-like life history that was morphologically dissimilar from previously known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behavior and prey range specificity, and characterized it with molecular phylogenetic methods and light and electron microscopy. The amoeba was a generalist predator (i.e. eukaryotroph), devouring a range of marine microalgae, with a strong affinity for some benthic diatoms and Chroomonas. Interestingly, the amoeba varied its feeding strategy depending on the prey species. Small diatoms were engulfed whole, while larger species were fed on through extraction with an invading pseudopodium. The SSU rRNA gene phylogenies robustly placed the amoeba in the most basal, poorly described lineage ("clade C") of the Vampyrellida. Based on the phylogenetic position and the distinct morphology of the studied amoeba, we here describe it as Sericomyxa perlucida gen. et sp. nov., and establish the new vampyrellid family Sericomyxidae for "clade C."

Particle shape does not affect ingestion and egestion of microplastics by the freshwater shrimp Neocaridina palmata.

The ingestion of microplastics (MPs) is well documented for various animals and spherical MPs (beads) in many studies. However, the retention time and egestion of MPs have been examined less, especially for irregular MPs (fragments) which are predominantly found in the environment. Furthermore, the accumulation of such particles in the gastrointestinal tract is likely to determine whether adverse effects are induced. To address this, we investigated if the ingestion and egestion of beads are different to those of fragments in the freshwater shrimp Neocaridina palmata. Therefore, organisms were exposed to 20-20,000 particles L-1 of either polyethylene (PE) beads (41 µm and 87 µm) or polyvinyl chloride (PVC) fragments (<63 µm). Moreover, shrimps were exposed to 20,000 particles L-1 of either 41 µm PE and 11 µm polystyrene (PS) beads or the PVC fragments for 24 h, followed by a post-exposure period of 4 h to analyze the excretion of particles. To simulate natural conditions, an additional fragment ingestion study was performed in the presence of food. After each treatment, the shrimps were analyzed for retained or excreted particles. Our results demonstrate that the ingestion of beads and fragments were concentration-dependent. Shrimps egested 59% of beads and 18% of fragments within 4 h. Particle shape did not significantly affect MP ingestion or egestion, but size was a relevant factor. Medium- and small-sized beads were frequently ingested. Furthermore, fragment uptake decreased slightly when co-exposed to food, but was not significantly different to the treatments without food. Finally, the investigations highlight that the assessment of ingestion and egestion rates can help to clarify whether MPs remain in specific organisms and, thereby, become a potential health threat.

A microbial eukaryote with a unique combination of purple bacteria and green algae as endosymbionts.

Oxygenic photosynthesizers (cyanobacteria and eukaryotic algae) have repeatedly become endosymbionts throughout evolution. In contrast, anoxygenic photosynthesizers (e.g., purple bacteria) are exceedingly rare as intracellular symbionts. Here, we report on the morphology, ultrastructure, lifestyle, and metagenome of the only "purple-green" eukaryote known. The ciliate Pseudoblepharisma tenue harbors green algae and hundreds of genetically reduced purple bacteria. The latter represent a new candidate species of the Chromatiaceae that lost known genes for sulfur dissimilation. The tripartite consortium is physiologically complex because of the versatile energy metabolism of each partner but appears to be ecologically specialized as it prefers hypoxic sediments. The emergent niche of this complex symbiosis is predicted to be a partial overlap of each partners' niches and may be largely defined by anoxygenic photosynthesis and possibly phagotrophy. This purple-green ciliate thus represents an extraordinary example of how symbiosis merges disparate physiologies and allows emergent consortia to create novel ecological niches.

Microplastics and sorbed contaminants - Trophic exposure in fish sensitive early life stages.

The present study evaluated very small microplastic particle (MPs) transfer to zebrafish and marine medaka larvae via prey experimentally exposed to MPs from the onset of feeding. Larvae were fed Paramecium or Artemia nauplii loaded with fluorescent 1-5 or 10-20 µm MP. Pollutant accumulation was analyzed by optically tracking of benzo[a]pyrene (BaP) and recording cyp1a transcription. Paramecium transferred 1-5 µm particles only, whereas Artemia efficiently transferred both MPs. Although zebrafish and medaka larvae fed from the onset of active food intake (2-3 dph, respectively) on Paramecium and from days 6-7 post-hatch on Artemia nauplii, neither MP accumulation nor translocation to tissues was detected. MP egestion started within few hours after ingestion. Cyp1a induction and fluorescent analyses proved BaP bioavailability after transfer via Paramecium and Artemia. Unicellular or plankton organisms ingest contaminants via MPS and transfer effectively these to sensitive early life-stages of vertebrates, giving rise to whole-life exposure.

External Validation of a Prognostic Score for Patients with Brain Metastases: Extended Diagnosis-Specific Graded Prognostic Assessment.

PURPOSE: The aim of our study was the external validation of an extended variant of the four-tiered diagnosis-specific graded prognostic assessment (DS-GPA) that includes more information about extracranial disease burden and blood test results, and predicts survival of patients with brain metastases. The extracranial DS-GPA (EC-GPA) includes serum albumin, lactate dehydrogenase, and number of extracranial organs involved. Originally, the score was developed in Germany. PATIENTS AND METHODS: A retrospective analysis of 236 patients with brain metastases treated with primary whole-brain radiotherapy in North-Norway was performed (independent external validation cohort). RESULTS: The four-tiered EC-GPA score showed good discrimination between all prognostic groups (log-rank test p < 0.05 for all pairwise comparisons). One-year survival was 0, 11, 30, and 100%, respectively. Median survival was 0.7 months (95% CI, 0.5-0.9) in the worst prognostic group, with a hazard ratio for death of 44.31 (95% CI, 5.78-339.50) compared to the best group. In the German database, the corresponding HR was 31.64 (median survival 0.4 months). The remaining hazard ratios in this validation study were 7.13 and 12.10, compared with 4.84 and 9.26 in the score development study. CONCLUSIONS: This study provides an independent validation of the EC-GPA, which was the best prognostic model for defining patients who did not benefit from radiation therapy of brain metastases in terms of overall survival in the original German study. The proposed modification of the established DS-GPA should undergo further validation in multi-institutional databases.

A natural toroidal microswimmer with a rotary eukaryotic flagellum.

We describe Idionectes vortex gen. nov., sp. nov., a unicellular microeukaryote that swims by continuous inversion of its surface, similar to a vortex ring. This previously unreported mode of motility approximates a hypothetical concept called the 'toroidal swimmer', in which a doughnut-shaped object rotates around its circular axis and travels in the opposite direction to its outer surface motion. During swimming, the flagellum of Idionectes rotates relative to its cell body, which is normally a hallmark of prokaryotic rather than eukaryotic flagella.

External validation of a prognostic score predicting overall survival for patients with brain metastases based on extracranial factors.

PURPOSE: The aim of our study was an external validation of the extracranial prognostic score predicting survival of patients with brain metastases receiving cranial irradiation on data from a single institution. MATERIALS AND METHODS: A retrospective analysis of 524 patients with brain metastases treated with cranial radiotherapy in a single tertiary center was performed. Three predictive scores were calculated and assessed for their ability to discriminate prognostic groups: (i) The Recursive Partitioning Analysis (RPA) score (available for 524 patients); (ii) the Diagnosis-Specific Graded Prognostic Assessment (DS-GPA) score (464 patients); (iii) the extracranial score (EC-S) developed by Nieder et al. which is based on serum albumin, lactate dehydrogenase (LDH) and the number of extracranial organs involved (157 patients). Discrimination of each score was assessed by Gönen & Heller's concordance probability estimate (CPE). The calibration was checked by comparing median survival estimates of each risk group with the corresponding values of the datasets from which the scores were derived. Finally, a multivariable Cox regression model was built by using the least absolute shrinkage and selection operator on a large number of variables including all three scores. RESULTS: With a CPE = 0.626 ±â€¯0.022, the EC-S had the best discriminatory power. The EC-S also appeared to be better calibrated and had the best ability to separate patients with a very poor prognosis: patients with combination of low albumin, elevated LDH and more than 1 extracranial organ with metastatic involvement had a median survival time of only 0.6 months (CI95% 0.1-1.1) and a hazard ratio for death of 6.36 (2.67-15.14) compared to patients with no extracranial metastases and normal levels of albumin and LDH. In the multivariable Cox model serum albumin, LDH, treatment modality, DS-GPA and EC-S were retained as prognostic factors. An ad hoc combination of both DS-GPA and EC-S into a new score was possible for 134 patients and indicated a slightly better discrimination (CPE = 0.636 ±â€¯0.023) than either DS-GPA or EC-S alone. CONCLUSIONS: This study provides an independent validation of the prognostic EC-S which was the best prognostic model for defining the patients who obviously did not benefit from radiation therapy of brain metastases in terms of overall survival. The combination of the EC-S with the established DS-GPA score resulted in a slight increase in discriminatory ability. The new EC-GPA score needs further validation in larger patient cohorts.