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The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.

Veterinarian competency in genetics is vital for a meaningful application of the rapidly growing number of genetic tests available for animals. We evaluated the use of genetic tests in the daily veterinary practice and the competency of university-employed veterinarians in applying basic principles of genetics in a clinical setting through an electronic survey with 14 cases and 7 statements on genetics. Ninety-one non-geneticist veterinarians from two veterinary faculties in two different countries responded. Almost half of the participants apply genetic tests during their daily work, with frequencies varying between weekly and once a year. The most common indication to request a genetic test was diagnostic testing of clinically ill patients. Although 80% of the veterinarians communicated the result of a genetic test themselves, only 56% of them found it “very to rather easy” to find the correct test, and only 32% of them always felt competent to interpret the result of the test. The number of correctly answered questions varied widely, with median scores of 9/14 (range: 0–14) and 5/7 (range: 0–7) for the cases and statements, respectively. Most difficulties were seen with recognition of pedigree inheritance patterns, while veterinarians scored better in breeding advice and probability of disease estimations. Veterinarians scored best on questions related to autosomal recessive inheritance, followed by complex, autosomal dominant, X-linked recessive, and X-linked dominant inheritance. This study exposed pain points in veterinarians’ knowledge and has led to the formulation of recommendations for future education and communication between laboratories, geneticists, and veterinarians.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi–Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3–6, and mutations similar to those found in patients with AGS cause autoinflammation7–12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7–10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

Acetaminophen (APAP) intoxication is the foremost cause of drug‐induced liver failure in developed countries. The only pharmacologic treatment option, N‐acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti‐MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP‐intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti‐MT antibodies. Anti‐MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS‐stimulated bone marrow‐derived macrophages. Importantly, NAC and anti‐MT antibodies were equally effective whereas administration of anti‐MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP‐induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono‐ or add‐on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization. Anti‐metallothionein antibody therapy reduces liver injury in experimental acetaminophen overdose.

Over the past decade, pharmacogenetics (PGx) became an essential tool for personalized medicine although its clinical implementation is still limited. We aimed to assess the current level of knowledge, applications, and expectations of Flemish pharmacists and physicians towards PGx and determine the factors that influence healthcare professionals’ knowledge of PGx, aiming to guide future implementation initiatives. A web-based cross-sectional survey was conducted from 8 March 2019 to 8 April 2019, targeting pharmacists, physicians, and trainees of both professions. Ten questions were used to assess the participants’ knowledge about PGx. Multivariable linear regression was used to assess the association of profession, experience, practice setting, and prior education with the level of PGx knowledge. In total, 201 Flemish healthcare providers participated, including 100 pharmacists, 73 physicians, and 28 trainees. The majority (78%) of participants were unfamiliar with the basic principles of PGx and its application in clinical practice. The mean percentage of correct answers achieved for the knowledge assessment questions was 34%. Only 9% had counseled patients, while 8% assisted other healthcare professionals on PGx tests the past year. Participants’ PGx knowledge was significantly affected by their profession, practice setting, and level of prior education independent of years of experience. These findings provide insight into factors affecting the knowledge of PGx and the current level of PGx implementation in Flemish clinical practice. This may form a basis for developing educational initiatives to enhance the clinical application of PGx in Flanders.

The development of TCRαβ and TCRγδ T cells comprises a step-wise process in which regulatory events control differentiation and lineage outcome. To clarify these mechanisms, we employed RNA-sequencing, ATAC–sequencing and ChIPmentation on well-defined thymocyte subsets that represent the continuum of human T cell development. The chromatin accessibility dynamics show clear stage specificity and reveal that human T cell-lineage commitment is marked by GATA3- and BCL11B-dependent closing of PU.1 sites. A temporary increase in H3K27me3 without open chromatin modifications is unique for β-selection, whereas emerging γδ T cells, which originate from common precursors of β-selected cells, show large chromatin accessibility changes due to strong T cell receptor (TCR) signaling. Furthermore, we unravel distinct chromatin landscapes between CD4+and CD8+αβ-lineage cells that support their effector functions and reveal gene-specific mechanisms that define mature T cells. This resource provides a framework for studying gene regulatory mechanisms that drive normal and malignant human T cell development.

Aim:This study provides clinicians and researchers with an informed choice between current commercially available targeted sequencing panels and exome sequencing panels in the context of pan-cancer pharmacogenetics. Materials & methods:Nine contemporary commercially available targeted pan-cancer panels and the xGen Exome Research Panel v2 were investigated to determine to what extent they cover the pharmacogenetic variant–drug interactions in five available cancer knowledgebases, and the driver mutations and fusion genes in the Cancer Genome Atlas. Results:xGen Exome Research Panel v2 and TrueSight Oncology 500 target 71.0 and 68.9% of the pharmacogenetic interactions in the available knowledgebases; and 93.7 and 86.0% of the driver mutations in the Cancer Genome Atlas, respectively. All other studied panels target lower percentages. Conclusion:Exome sequencing outperforms pan-cancer targeted sequencing panels in terms of covered cancer pharmacogenetic variant–drug interactions and pharmacogenetic cancer variants.

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation–sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation–sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.

Clinical, pathological, and genetic findings of a primary hereditary ataxia found in a Malinois dog family are described and compared with its human counterpart. Based on the family history and the phenotype/genotype relationships already described in humans and dogs, a causal variant was expected to be found in KCNJ10. Rather surprisingly, whole-exome sequencing identified the SLC12A6NC_006612.3(XM_014109414.2): c.178_181delinsCATCTCACTCAT (p.(Met60Hisfs*14)) truncating variant. This loss-of-function variant perfectly segregated within the affected Malinois family in an autosomal recessive way and was not found in 562 additional reference dogs from 18 different breeds, including Malinois. In humans, SLC12A6variants cause “agenesis of the corpus callosum with peripheral neuropathy” (ACCPN, alias Andermann syndrome), owing to a dysfunction of this K+–Cl−cotransporter. However, depending on the variant (including truncating variants), different clinical features are observed within ACCPN. The variant in dogs encodes the shortest isoform described so far and its resultant phenotype is quite different from humans, as no signs of peripheral neuropathy, agenesis of the corpus callosum nor obvious mental retardation have been observed in dogs. On the other hand, progressive spinocerebellar ataxia, which is the most important feature of the canine phenotype, hindlimb paresis, and myokymia-like muscle contractions have not been described in humans with ACCPN so far. As this is the first report of a naturally occurring disease-causing SLC12A6variant in a non-human species, the canine model will be highly valuable to better understand the complex molecular pathophysiology of SLC12A6-related neurological disorders and to evaluate novel treatment strategies.

Over the past decade, significant advances have been made to unravel molecular mechanisms of stress response in different ecotoxicological model species. Within this study, we focus on population level transcriptomic responses of a natural population of Daphnia magnaStraus, (1820), to heavy metals. We aim to characterize the population level transcriptomic responses, which include standing genetic variation, and improve our understanding on how populations respond to environmental stress at a molecular level. We studied population level responses to two heavy metals, copper and arsenic, and their binary mixture across time. Transcriptomic patterns identified significantly regulated gene families and genes at the population level including cuticle proteins and resilins. Furthermore, some of these differentially regulated gene families, such as cuticle proteins, were also significantly enriched for genetic variations including SNPs and MNPs. In general, genetic variation was observed in specific gene families, many of which are known to be involved in stress response. Overall, our results indicate that molecular stress responses can be identified within natural populations and that linking molecular mechanisms with genetic variation at the population level could contribute significantly to adverse outcome frameworks.

Epigenetic mechanisms have been found to play important roles in environmental stress response and regulation. These can, theoretically, be transmitted to future unexposed generations, yet few studies have shown persisting stress-induced transgenerational effects, particularly in invertebrates. Here, we focus on the aquatic microcrustacean Daphnia,a parthenogenetic model species, and its response to salinity stress. Salinity is a serious threat to freshwater ecosystems and a relevant form of environmental perturbation affecting freshwater ecosystems. We exposed one generation of D. magnato high levels of salinity (F0) and found that the exposure provoked specific methylation patterns that were transferred to the three consequent nonexposed generations (F1, F2, and F3). This was the case for the hypomethylation of six protein-coding genes with important roles in the organisms’ response to environmental change: DNA damage repair, cytoskeleton organization, and protein synthesis. This suggests that epigenetic changes in Daphniaare particularly targeted to genes involved in coping with general cellular stress responses. Our results highlight that epigenetic marks are affected by environmental stressors and can be transferred to subsequent unexposed generations. Epigenetic marks could therefore prove to be useful indicators of past or historic pollution in this parthenogenetic model system. Furthermore, no life history costs seem to be associated with the maintenance of hypomethylation across unexposed generations in Daphniafollowing a single stress exposure.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Epigenetic changes can be studied with an untargeted characterization of histone post-translational modifications (PTMs) by liquid chromatography–mass spectrometry (LC–MS). While prior information about more than 20 types of histone PTMs exists, little is known about histone PTM combinations (PTMCs). Because of the combinatorial explosion it is intrinsically impossible to consider all potential PTMCs in a database search. Consequentially, high-scoring false positives with unconsidered but correct alternative isobaric PTMCs can occur. Current quality controls can neither estimate the amount of unconsidered alternatives nor flag potential false positives. Here, we propose a conceptual workflow that provides such options. In this workflow, an in silico modeling of all candidate isoforms with known-to-exist PTMs is made. The most frequently occurring PTM sets of these candidate isoforms are determined and used in several database searches. This suppresses the combinatorial explosion while considering as many candidate isoforms as possible. Finally, annotations can be classified as unique or ambiguous, the latter implying false positives. This workflow was evaluated on an LC–MS data set containing 44 histone extracts. We were able to consider 60% of all candidate isoforms. Importantly, 40% of all annotations were classified as ambiguous. This highlights the need for a more thorough evaluation of modified peptide annotations.

In the human thymus, a CD10+ PD-1+ TCRαβ+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8β expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny.

ABSTRACTHelicobacter heilmanniinaturally colonizes the stomachs of dogs and cats and has been associated with gastric disorders in humans. Nine feline Helicobacterstrains, classified as H. heilmanniibased on ureABand 16S rRNA gene sequences, were divided into a highly virulent and a low-virulence group. The genomes of these strains were sequenced to investigate their phylogenetic relationships, to define their gene content and diversity, and to determine if the differences in pathogenicity were associated with the presence or absence of potential virulence genes. The capacities of these helicobacters to bind to the gastric mucosa were investigated as well. Our analyses revealed that the low-virulence strains do not belong to the species H. heilmanniibut to a novel, closely related species for which we propose the name Helicobacter ailurogastricus. Several homologs of H. pylorivirulence factors, such as IceA1, HrgA, and jhp0562-like glycosyltransferase, are present in H. heilmanniibut absent in H. ailurogastricus. Both species contain a VacA-like autotransporter, for which the passenger domain is remarkably larger in H. ailurogastricusthan in H. heilmannii. In addition, H. ailurogastricusshows clear differences in binding to the gastric mucosa compared to H. heilmannii. These findings highlight the low-virulence character of this novel Helicobacterspecies.

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks the first time that isobaric tags have been applied for the large-scale analysis of phosphopeptides. Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of Phospho-iTRAQ with regards to the experimental setup, the proteome coverage was extended through gel fractionation, while an internal replicate measurement created more stringent data analysis opportunities. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data have been deposited to the ProteomeXchange with identifier PXD001574.

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.

Obsessivecompulsive disorder OCD is a severe anxiety disorder characterized by obsessions and compulsions. The core symptom of OCD is compulsivity, the inability to stop thinking or acting when you want to, despite being aware of the uselessness of the content or the adverse consequences. To initiate a systematic search for genetic mechanisms underlying the pathophysiology of compulsivity, a panel of chromosome substitution CS strains, derived from mice that suppress C57BL6J strain or maintain AJ strain high levels of repetitive wheel running during 2 hr of daily limited food access, was screened for this compulsive behavior. Following the genetic screen, we found linkage between compulsive wheel running and mouse chromosomes 2, 6, and 7 that show overlap with recently identified human linkage regions for OCD on chromosomes 7p and 15q. In the overlapping humanmouse genomic region, the CRH receptor 2 CRHR2 gene was tested in a human case–control study. An initial exploration in OCD cases versus controls failed to detect an association between fourcandidate CRH2R SNPs within this homologous linkage region and OCD. Genetic fine mapping of compulsivity in mice provides new opportunities to reveal mechanisms underlying this significant psychiatric trait. © 2009 WileyLiss, Inc.

ObsessiveCompulsive Disorder OCD is characterized by the presence of obsessions and compulsions, and shows considerable phenotypic variability. Family and twin studies have indicated a genetic component in the etiology of OCD, and the catecholOmethyl transferase COMT gene is an important candidate gene for OCD. This study investigates the influence of the functional COMT Val158Metpolymorphism on the phenotypic expression of OCD, using an itemlevel factoranalytic approach in a large sample. The COMT Val158Metvariant was genotyped in 373 patients and 462 controls. It was tested whether there was an association between the COMT Val158Metpolymorphism and OCD or dimensional phenotypes such as YBOCS severity score, age of onset of obsessivecompulsive symptoms and six symptom dimensions recently found in a large itemlevel factoranalytic study Katerberg et al., submitted. We further investigated possible sexspecific associations between the COMT Val158Metpolymorphism and OCD or dimensional phenotypes. There was a trend for an association of the COMT 158Metallele with OCD in males, and an interaction between the COMT Val158Metgenotype and sex on the somatic and sensory phenomena symptom dimension, with females showing lower scores. In conclusion, a dimensional approach seems fruitful in detecting genes of importance for OCD. © 2009 WileyLiss, Inc.

•Small RNAs are present in cell-free locust hemolymph•MicroRNAs circulate in a stable form in locust hemolymph serum•The abundances of microRNAs differ between sera of young and old locusts•Modulating the extracellular levels of two specific microRNAs affects the moulting process

Loss of function of adenosine deaminase acting on double-stranded RNA (dsRNA)-1 (ADAR1) causes the severe autoinflammatory disease Aicardi-Goutières syndrome (AGS). ADAR1 converts adenosines into inosines within dsRNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and the poorly defined Z conformation (Z-RNA). Mutations in the Z-RNA-binding Zα domain of ADAR1 are common in patients with AGS. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. We demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by base pairing of inversely oriented short interspersed nuclear elements. Preventing ADAR1 binding to Z-RNA triggers an MDA5/MAVS-mediated type I interferon response and leads to the development of lethal autoinflammation in mice. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.

Veterinarian competency in genetics is vital for a meaningful application of the rapidly growing number of genetic tests available for animals. We evaluated the use of genetic tests in the daily veterinary practice and the competency of university-employed veterinarians in applying basic principles of genetics in a clinical setting through an electronic survey with 14 cases and 7 statements on genetics. Ninety-one non-geneticist veterinarians from two veterinary faculties in two different countries responded. Almost half of the participants apply genetic tests during their daily work, with frequencies varying between weekly and once a year. The most common indication to request a genetic test was diagnostic testing of clinically ill patients. Although 80% of the veterinarians communicated the result of a genetic test themselves, only 56% of them found it “very to rather easy” to find the correct test, and only 32% of them always felt competent to interpret the result of the test. The number of correctly answered questions varied widely, with median scores of 9/14 (range 0–14) and 5/7 (range 0–7) for the cases and statements, respectively. Most difficulties were seen with recognition of pedigree inheritance patterns, while veterinarians scored better in breeding advice and probability of disease estimations. Veterinarians scored best on questions related to autosomal recessive inheritance, followed by complex, autosomal dominant, X-linked recessive, and X-linked dominant inheritance. This study exposed pain points in veterinarians’ knowledge and has led to the formulation of recommendations for future education and communication between laboratories, geneticists, and veterinarians.

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance.

Even though radiography is one of the most frequently used imaging techniques for orthopaedic disorders, it has been demonstrated that the interpretation can vary between assessors. As such, the purpose of this study was to examine the intraobserver and interobserver agreement and the influence of level of expertise on the interpretation of radiographs of the stifle in dogs with and without cranial cruciate ligament rupture (CCLR). Sixteen observers, divided in four groups according to their level of experience, evaluated 30 radiographs (15 cases with CCLR and 15 control stifles) twice. Each observer was asked to evaluate joint effusion, presence and location of degenerative joint disease, joint instability and whether CCLR was present or absent. Overall, intraobserver and interobserver agreement ranged from fair to almost perfect with a trend towards increased agreement for more experienced observers. Additionally, it was found that stifles that were classified with high agreement have either overt disease characteristics or no disease characteristics at all, in comparison to the ones that are classified with a low agreement. Overall, the agreement on radiographic interpretation of CCLR was high, which is important, as it is the basis of a correct diagnosis and treatment.

No relevant conflicts of interest to declare.

ABSTRACTThe enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection.IMPORTANCERecent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family.

Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine differentially expressed genes (DEGs) (Pvalue ≤0.05 and absolute fold change ≥2) and to provide functional interpretation. A total of 10 743 transcripts were identified and 253 genes were found to be upregulated and 108 to be downregulated in the pregnant ipsilateral oviduct when compared to the cyclic ipsilateral oviduct. Comparison of the ipsilateral and the contralateral oviduct indicated 164 DEGs in pregnant mares and 77 DEGs in cyclic mares. Enriched functional categories were detected only in the comparison of pregnant and cyclic ipsilateral oviducts and showed that the equine embryo affects the expression of immune response-related genes in the oviduct, with marked upregulation of interferon-associated genes. This research represents the foundation for further assessment of the role of specific genes in the early embryo-maternal dialogue of the horse.

Emerging evidence suggests that long non-coding RNAs (lncRNAs) are critically involved in a variety of human tumor entities and the identification of cancer-associated lncRNAs might reveal new prognostic biomarkers or novel therapeutic targets for the treatment of human cancer. In this study, we characterized the lncRNA expression signature associated with ETV6/RUNX1-positive pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), one of the most prevalent genetic subtypes of childhood leukemia.