Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer, and in this process, some "old" biomarkers are reutilized and given a new purpose. It is not always recognized that by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory-developed tests (LDTs) of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays with 32 cases and performed their in-house Ki-67 assays. The results were assessed using QuPath, an open-source software application for bioimage analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20%, and 30% cutoffs. Overall, PPA and NPA varied depending on the selected cutoff; participants were more successful with 5% and 10%, than with 20% and 30% cutoffs. Only 4 of 16 laboratories had robust IHC protocols with acceptable PPA for all cutoffs. The lowest PPA for the 5% cutoff was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cutoff was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cutoffs. The poor agreement was not due to the readout but rather due to IHC protocol conditions. International Ki-67 in Breast Cancer Working Group (IKWG) recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Pediatric anesthesiology in Brazil, Chile, and Mexico.

BACKGROUND: Latin America comprises an extensive and diverse territory composed of 33 countries in the Caribbean, Central, and South America where Romance languages-languages derived from Latin are predominantly spoken. Economic disparities exist, with inequitable access to pediatric surgical care. The Latin American Surgical Outcomes Study in Pediatrics (LASOS-Peds), a multi-national collaboration, will determine safety of pediatric anesthesia and perioperative care. OBJECTIVE: Below, we provide a descriptive initiative to share how pediatric anesthesia in Brazil, Chile, and Mexico operate. Theses descriptions do not represent all of Latin America. DESCRIPTIONS AND CONCLUSIONS: Brazil an upper middle-income country, population 203 million, has a public system insufficiently resourced and a private system, resulting in inequitable safety and accessibility. Surgical complications constitute the third leading cause of mortality. Anesthesiology residency is 3 years, with required rotations in pediatric anesthesia; five hospitals offer pediatric anesthesia fellowships. Anesthesiology is a physician-only practice. A Pediatric Anesthesia Committee within the Brazilian Society of Anesthesiology offers education through seasonal courses and workshops including pediatric advanced life support. Chile is a high-income country, population 19.5 million, the majority cared for in the public system, the remainder in university, private, or military systems. Government efforts have gradually corrected the long-standing anesthesiology shortage: twenty 3-year residency programs prepare graduates for routine pediatric cases. The Chilean Society of Anesthesiology runs a 1-month program for general anesthesiologists to enhance pediatric anesthesia skills. Pediatric anesthesia fellowship training occurs in Europe, USA, and Australia, or in two 2-year Chilean university programs. Public health policies have increased the medical and surgical pediatric specialists and general anesthesiologists, but not pediatric anesthesiologists, which creates safety concerns for neonates, infants, and medically complex. Chile needs more pediatric anesthesia fellowship programs. Mexico, an upper middle-income country, with a population of about 126 million, has a five-sector healthcare system: public, social security for union workers, state for public employees, armed forces for the military, and a private "self-pay." There are inequities in safety and accessibility for children. Pediatric Anesthesiology fellowship is 2 years, after 3 years residency. A shortage of pediatric anesthesiologists limits accessibility and safety for surgical care, driven by added training at low salary and hospital under appreciation. The Mexican Society of Pediatric Anesthesiology conducts refresher courses, workshops, and case conferences. Insufficient resources and culture limits research.

The contribution of inflammatory astrocytes to BBB impairments in a brain-chip model of Parkinson's disease.

Astrocyte dysfunction has previously been linked to multiple neurodegenerative disorders including Parkinson's disease (PD). Among their many roles, astrocytes are mediators of the brain immune response, and astrocyte reactivity is a pathological feature of PD. They are also involved in the formation and maintenance of the blood-brain barrier (BBB), but barrier integrity is compromised in people with PD. This study focuses on an unexplored area of PD pathogenesis by characterizing the interplay between astrocytes, inflammation and BBB integrity, and by combining patient-derived induced pluripotent stem cells with microfluidic technologies to generate a 3D human BBB chip. Here we report that astrocytes derived from female donors harboring the PD-related LRRK2 G2019S mutation are pro-inflammatory and fail to support the formation of a functional capillary in vitro. We show that inhibition of MEK1/2 signaling attenuates the inflammatory profile of mutant astrocytes and rescues BBB formation, providing insights into mechanisms regulating barrier integrity in PD. Lastly, we confirm that vascular changes are also observed in the human postmortem substantia nigra of both males and females with PD.

Denoising digital breast tomosynthesis projections using deep learning with synthetic data as training set.

Purpose: Image denoising based on deep neural networks (DNN) needs a big dataset containing digital breast tomosynthesis (DBT) projections acquired in different radiation doses to be trained, which is impracticable. Therefore, we propose extensively investigating the use of synthetic data generated by software for training DNNs to denoise DBT real data. Approach: The approach consists of generating a synthetic dataset representative of the DBT sample space by software, containing noisy and original images. Synthetic data were generated in two different ways: (a) virtual DBT projections generated by OpenVCT and (b) noisy images synthesized from photography regarding noise models used in DBT (e.g., Poisson-Gaussian noise). Then, DNN-based denoising techniques were trained using a synthetic dataset and tested for denoising physical DBT data. Results were evaluated in quantitative (PSNR and SSIM measures) and qualitative (visual analysis) terms. Furthermore, a dimensionality reduction technique (t-SNE) was used for visualization of sample spaces of synthetic and real datasets. Results: The experiments showed that training DNN models with synthetic data could denoise DBT real data, achieving competitive results to traditional methods in quantitative terms but showing a better balance between noise filtering and detail preservation in a visual analysis. T-SNE enables us to visualize if synthetic and real noises are in the same sample space. Conclusion: We propose a solution for the lack of suitable training data to train DNN models for denoising DBT projections, showing that we just need the synthesized noise to be in the same sample space as the target image.

Mentalizing, Resilience, and Mental Health Status among Healthcare Workers during the COVID-19 Pandemic: A Cross-Sectional Study.

The COVID-19 pandemic has caused unprecedented stress on healthcare professionals worldwide. Since resilience and mentalizing capacity play very important preventive roles when it comes to mental health, the main goal of this study was to determine whether the capacity for mentalizing and resilience could explain the levels of depression, anxiety, and stress among healthcare workers during the COVID-19 pandemic. The study was conducted in Serbia on a sample of 406 healthcare workers (141 doctors and 265 nurses) aged 19 to 65 (M = 40.11, SD = 9.41). The participants' mental health status was evaluated using the Depression, Anxiety, and Stress Scale-DASS-42. The Reflective Functioning Questionnaire was used to evaluate the capacity for mentalizing. Resilience was assessed using the Brief Resilience Scale. The results of the correlation analysis showed that there were negative correlations between resilience and all three dimensions of mental health status: depression, anxiety, and stress. Hypermentalizing was negatively correlated with depression, anxiety, and stress, while hypomentalizing was positively correlated. Hierarchical linear regression analysis showed that both resilience and hypermentalizing were significant negative predictors of depression, anxiety, and stress, and that hypomentalizing was a significant positive predictor of depression, anxiety, and stress. Furthermore, socioeconomic status was a significant negative predictor of depression, anxiety, and stress. Marital status, number of children, and work environment were not statistically significant predictors of any of the three dimensions of mental health status among the healthcare workers in this study. There is an urgent need to establish and implement strategies to foster resilience and enhance the capacity for mentalizing among healthcare workers in order to minimize the devastating effects of the COVID-19 pandemic on mental health.

CamNuvem: A Robbery Dataset for Video Anomaly Detection.

(1) Background: The research area of video surveillance anomaly detection aims to automatically detect the moment when a video surveillance camera captures something that does not fit the normal pattern. This is a difficult task, but it is important to automate, improve, and lower the cost of the detection of crimes and other accidents. The UCF-Crime dataset is currently the most realistic crime dataset, and it contains hundreds of videos distributed in several categories; it includes a robbery category, which contains videos of people stealing material goods using violence, but this category only includes a few videos. (2) Methods: This work focuses only on the robbery category, presenting a new weakly labelled dataset that contains 486 new real-world robbery surveillance videos acquired from public sources. (3) Results: We have modified and applied three state-of-the-art video surveillance anomaly detection methods to create a benchmark for future studies. We showed that in the best scenario, taking into account only the anomaly videos in our dataset, the best method achieved an AUC of 66.35%. When all anomaly and normal videos were taken into account, the best method achieved an AUC of 88.75%. (4) Conclusion: This result shows that there is a huge research opportunity to create new methods and approaches that can improve robbery detection in video surveillance.

Hydroxy-selenomethionine as an organic source of selenium in the diet improves boar reproductive performance in artificial insemination programs.

This study aimed to compare different selenium (Se) sources in the diet on boar's semen quality and fertility. For this, 28 boars aged 8 to 28 mo were fed with the following dietary treatments for 95 d: 0.3 mg Se/kg as sodium selenite (SS; n = 14) and 0.3 mg Se/kg as hydroxy-selenomethionine (OH-SeMet; n = 14). During this period, two experiments were carried out. In experiment 1, the semen of all boars was evaluated every 2 wk. Raw semen was initially evaluated for the processing of seminal doses, which were stored at 17 °C for 72 h, followed by sperm quality assessments. Furthermore, Se concentration and glutathione peroxidase (GPx) activity were measured in the seminal plasma. In experiment 2, 728 females were inseminated weekly with seminal doses from boars of the different experimental groups to further assess in vivo fertility and litter characteristics. Results demonstrated that boars fed OH-SeMet had more Se in their seminal plasma (P < 0.05), showing the greater bioavailability of the organic source in the male reproductive system. Moreover, boars fed OH-SeMet tended (P < 0.10) toward a higher total sperm count in the ejaculate (66.60 vs. 56.57 × 109 sperm) and the number of seminal doses (22.11 vs. 18.86; 3 × 109 sperm/dose) when compared with those fed SS. No effect of the dietary treatments was observed on GPx activity in seminal plasma (P > 0.05) as well as on raw and stored semen quality (P > 0.05). Under in vivo conditions, seminal doses from boars fed OH-SeMet tended (P < 0.10) toward a higher pregnancy rate at weeks 3, 5, and 8, and also resulted in a higher (P < 0.05) percentage of pregnant females in the overall period (99.30 vs. 97.00). In conclusion, the replacement of SS with OH-SeMet in boars' diet can improve sperm production and results in better reproductive performance for them, bringing greater productivity and profitability to artificial insemination centers and commercial pig farms.

Neoadjuvant Chemotherapy in Breast Cancer: Review of the Evidence and Conditions That Facilitated Its Use during the Global Pandemic.

Practice and behaviour change in healthcare is complex, and requires a set of critical steps that would be needed to implement and sustain the change. Neoadjuvant chemotherapy for breast cancer is traditionally used for locally advanced disease and is primarily advantageous for surgical downstaging purposes. However, it does also offer patients with certain biologic subtypes such as the triple negative or Her2 positive breast cancers the opportunity to improve survival, even in early stage disease. During the height of the pandemic, an opportunity and motivation for the increased use of neoadjuvant therapy in breast cancer was identified. This paper describes the conditions that have supported this practice change at the provider and institutional levels. We also include our own institutional algorithm based on tumor biology and extent of disease that have guided our decisions on breast cancer management during the pandemic. Our processes can be adapted by other institutions and breast oncology practices in accordance with local conditions and resources, during and beyond the pandemic.

The Function and Evolution of Motile DNA Replication Systems in Ciliates.

DNA replication is a ubiquitous and conserved cellular process. However, regulation of DNA replication is only understood in a small fraction of organisms that poorly represent the diversity of genetic systems in nature. Here we used computational and experimental approaches to examine the function and evolution of one such system, the replication band (RB) in spirotrich ciliates, which is a localized, motile hub that traverses the macronucleus while replicating DNA. We show that the RB can take unique forms in different species, from polar bands to a "replication envelope," where replication initiates at the nuclear periphery before advancing inward. Furthermore, we identify genes involved in cellular transport, including calcium transporters and cytoskeletal regulators, that are associated with the RB and may be involved in its function and translocation. These findings highlight the evolution and diversity of DNA replication systems and provide insights into the regulation of nuclear organization and processes.

Activation of TRPA1 nociceptor promotes systemic adult mammalian skin regeneration.

Adult mammalian wounds, with rare exception, heal with fibrotic scars that severely disrupt tissue architecture and function. Regenerative medicine seeks methods to avoid scar formation and restore the original tissue structures. We show in three adult mouse models that pharmacologic activation of the nociceptor TRPA1 on cutaneous sensory neurons reduces scar formation and can also promote tissue regeneration. Local activation of TRPA1 induces tissue regeneration on distant untreated areas of injury, demonstrating a systemic effect. Activated TRPA1 stimulates local production of interleukin-23 (IL-23) by dermal dendritic cells, leading to activation of circulating dermal IL-17-producing γδ T cells. Genetic ablation of TRPA1, IL-23, dermal dendritic cells, or γδ T cells prevents TRPA1-mediated tissue regeneration. These results reveal a cutaneous neuroimmune-regeneration cascade triggered by topical TRPA1 activators that promotes adult mammalian tissue regeneration, presenting a new avenue for research and development of therapies for wounds and scars.

Exhaled nitric oxide measurement before pediatric adenotonsillectomy: A feasibility study.

BACKGROUND: Exhaled nitric oxide (eNO) is a known biomarker for the diagnosis and monitoring of bronchial hyperreactivity in adults and children. AIMS: To investigate the potential role of eNO measurement for predicting perioperative respiratory adverse events in children, we sought to determine its feasibility and acceptability before adenotonsillectomy. METHODS: We attempted eNO testing in children, 4-12 years of age, immediately prior to admission for outpatient adenotonsillectomy. We used correlations between eNO levels and postoperative adverse respiratory events to make sample size predictions for future studies that address the predictability of the device. RESULTS: One hundred and three (53%) of 192 children were able to provide an eNO sample. The success rate increased with age from 23% (9%-38%) at age 4 to over 85% (54%-98%) after age 9. Using the eNO normal value (<20 ppb) as a cutoff, an expected sample size to detect a significant difference between children with and without adverse events is 868, assuming that respiratory adverse events occur in 29% of children. CONCLUSIONS: eNO testing on the day of surgery has limited feasibility in children younger than 7 years of age. The most common reason for failure was inadequate physical performance while interacting with the testing device. The role of this respiratory biomarker in the context of perioperative outcomes for pediatric adenotonsillectomy remains unknown and should be further studied with improved technologies.

Selecting Patients for Oncotype DX Testing Using Standard Clinicopathologic Information.

INTRODUCTION: Indiscriminate ordering of Oncotype DX (ODX) is expensive and of poor value to patients, physicians, and health care providers. The 3 Magee equations, Gage Algorithm, and University of Tennessee predictive algorithm all use standard clinicopathologic data to provide surrogate ODX scores. In this hypothesis-generating study, we evaluated whether these prognostic scores could be used to identify patients unlikely to benefit from additional ODX testing. PATIENTS AND METHODS: Retrospective data was collected from 302 patients with invasive ductal breast cancer and available ODX scores. Additional data was available for: Magee equations 1 (212 patients), 2 (299 patients), 3 (212 patients), Gage Algorithm (299 patients), and University of Tennessee predictive algorithm (286 patients). ODX scores were banded according to the TAILORx results. RESULTS: Correlation with ODX scores was between 0.7 and 0.8 (Gage), 0.8 and 0.9 (Magee 2, University of Tennessee predictive algorithm), and > 0.9 (Magee 1 and 3). Magee 3 was the most robust and is proposed as a screening tool: for patients aged ≤ 50 years, ODX testing would be not required if the Magee 3 score was < 14 or ≥ 20; for those aged > 50 years, ODX would not be required if the Magee 3 score was < 18 or ≥ 26. Using these cut-offs, 110 (51.9%) of 212 patients would be deemed as not requiring ODX testing, and 109 (99.1%) of110 patients would be appropriately managed. CONCLUSIONS: Use of all formulae, and the Magee 3 equation in particular, are proposed as possible screening tools for ODX testing, resulting in significantly reduced frequency of ODX testing. This requires validation in other populations.

Celebrating 75 years.

INTRODUCTION: Following the introduction of shock wave lithotripsy (SWL), ureteroscopy (URS), and percutaneous nephrolithotomy (PCNL), the subspecialty of endourology was born in the late 1970s. The purpose of this study was to report milestones in Canadian endourology, highlighting Canada's contributions to the field. METHODS: A review of the literature was performed from the late 1970s to the present. The literature review included bibliographic and digital resources. Additionally, records and recollections by various individuals were used, including some who were directly involved. RESULTS: Endourology was born in Canada when SWL, URS, and PCNL emerged as minimally invasive treatment options for stones in the early to mid-1980s. According to our research, the first PCNL was performed at the University of Toronto in 1981. Dr. Joachim Burhenne, a Harvard-trained radiologist from Germany, first used extracorporeal SWL in Canada at the University of British Columbia (UBC) for the treatment of biliary stones. Treatment for urinary tract stones followed at UBC and Dalhousie University. The first worldwide use of the holmium laser for lithotripsy of urinary tract calculi took place at the University of Western Ontario. Other endourology milestones in Canada include the formation of the Canadian Endourology Group and the emergence of the Endourological Society-accredited fellowship programs at the University of Toronto and Western University in the 1990s. Canada hosted the 21st and 35th World Congress of Endourology and Shock Wave Lithotripsy annual meeting in Montreal and Vancouver, respectively. CONCLUSIONS: Canadian urologists have led many advances in SWL, URS, and PCNL over the past four decades and, for a relatively small community, have made significant contributions to the field. Through the training of the next generation of endourologists at Canadian institutions, the future of endourology in Canada is bright.

Secondhand Smoke exposure and risk of Obstructive Sleep Apnea in Children.

OBJECTIVES: Obstructive sleep apnea (OSA) has a prevalence of 4% in children. Few studies have explored the role of secondhand smoke (SHS) on OSA severity and have shown contradicting results. Most studies have focused on the effect of SHS on snoring. This study explored the association of SHS exposure and OSA severity in children aged 3-18 years. METHODS: This is a retrospective single center IRB-approved study. Electronic Medical Records (EMR) were queried between 1/24/2015 and 1/24/2018 to obtain data on SHS exposure with standard questionnaires from perioperative database. SHS was analyzed as a binary variable and OSA was measured using obstructive apnea hypopnea index (OAHI) from polysomnography (PSG) as a continuous variable. Analyses were done on all children and in those with severe OSA (OAHI≥10/h) as a subgroup. RESULTS: EMR query yielded 101,884 children of whom 3776 had PSG. Limiting baseline PSG in 3-18-year-old and reliable information on SHS yielded 167 analyzable children of whom 70 had severe OSA. Children exposed to SHS had significantly more public insurance than non-exposed (p < 0.0001). Among children with severe OSA, median OAHI was significantly higher in SHS exposed compared to non-exposed (29.0vs.19.5,p = 0.04), but not across all children. In multivariable analysis SHS exposure increased OAHI by 48% in severe OSA subgroup (95%CI: 8%-102%; p = 0.01) when adjusted for race, body mass index, and adjusted household income. CONCLUSION: Children aged 3-18 years with severe OSA who were exposed to SHS were found to have 1.48 increase in odds of OAHI than those without SHS exposure. Results could be limited by retrospective nature of study and EMR tools.

Cysteamine as a novel disease-modifying compound for Parkinson's disease: Over a decade of research supporting a clinical trial.

To date, medical and surgical interventions offered to patients with Parkinson's disease (PD) serve only to manage clinical symptoms; they have not shown the capacity to halt nor reverse degenerative processes. There is therefore an urgent need to identify and/or develop therapeutic strategies that will demonstrate 'disease modifying' capacities. The molecule cystamine, and its reduced form cysteamine, act via a number of pathways determined to be critical to the pathogenesis of PD. In particular, cystamine is capable of crossing the blood-brain barrier, and both agents (cystamine and cysteamine) can promote the secretion of neurotrophic factors, inhibit oxidative stress, reduce inflammatory responses and importantly, have already been trialed in humans for a number of other clinical indications. In the last decade, our laboratory has accumulated compelling evidence that both cystamine and cysteamine can halt, and even reverse, ongoing neurodegenerative processes in a number of different models of PD, and as such, should now be taken forward to clinical trials in PD.

STBUR: Sleep trouble breathing and unrefreshed questionnaire: Evaluation of screening tool for postanesthesia care and disposition.

BACKGROUND: The Snoring, Trouble Breathing, and Un-Refreshed (STBUR) questionnaire is a five-question screening tool for pediatric sleep-disordered breathing and risk for perioperative respiratory adverse events in children. The utility of this questionnaire as a preoperative risk-stratification tool has not been investigated. In view of limited availability of screening tools for preoperative pediatric sleep-disordered breathing, we evaluated the questionnaire's performance for postanesthesia adverse events that can impact postanesthesia care and disposition. METHODS: The retrospective study protocol was approved by the institutional research board. The data were analyzed using two different definitions for a positive screening based on a five-point scale: low threshold (scores 1 to 5) and high threshold (score of 5). The primary outcome was based on the following criteria: (a) supplemental oxygen therapy following postanesthesia care unit (PACU) stay until hospital discharge, (b) greater than two hours during phase 1 recovery, (c) anesthesia emergency activation in the PACU, and (d) unplanned hospital admission. RESULTS: About 6025 patients completed the questionnaire during the preoperative evaluation. And 1522 patients had a low threshold score and 270 had a high-threshold score. We found statistically significant associations in three outcomes based on the low threshold score: supplemental oxygen therapy (negative-predictive value [NPV] 0.97, 95% CI 0.97-98), PACU recovery time (NPV 0.99, 95% CI 0.99-0.99) and escalation of care (NPV 0.98, 95% CI 0.97-0.98). Positive-predictive values were statistically significant for all outcomes except anesthesia emergency in the PACU. CONCLUSION: The Snoring, Trouble Breathing, and Un-Refreshed questionnaire identified patients at higher risk for prolonged phase 1 recovery, oxygen therapy requirement, and escalation of care. The questionnaire's high-negative predictive value and specificity may make it useful as a screening tool to identify patients at low risk for prolonged stay in PACU.

The Sanguicolous Apostome Metacollinia luciensis Jankowski 1980 (Colliniidae, Apostomatia, Ciliophora) Is Not Closely Related to Other Sanguicolous Apostomes.

The apostome family Colliniidae includes species that are adapted to the hemocoel/blood of various invertebrates, particularly crustaceans. To explore the phylogeny of these sanguicolous apostomes, Metacollinia luciensis was collected in August 2015 at Roscoff from the amphipod host, Orchestia gammarellus. Ciliates were Protargol stained and DNA was extracted. The small subunit rRNA (SSUrRNA) and cytochrome c oxidase subunit I (cox1) genes were amplified. Molecular phylogenetic analyses of the SSUrRNA genes unambiguously grouped M. luciensis with other apostomes with robust bootstrap support, but separated it distinctly from the pseudocolliniid clade. While there are only cox1 sequences for a subset of these apostomes, M. luciensis was also distant from the pseudocolliniids and separated from them by species of the exuviotrophic apostome Hyalophysa. These results confirm the distinctness of the families Colliniidae and Pseudocolliniidae.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.