CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Histone Chaperone Deficiency in Arabidopsis Plants Triggers Adaptive Epigenetic Changes in Histone Variants and Modifications.

At the molecular scale, adaptive advantages during plant growth and development rely on modulation of gene expression, primarily provided by epigenetic machinery. One crucial part of this machinery is histone posttranslational modifications, which form a flexible system, driving transient changes in chromatin, and defining particular epigenetic states. Posttranslational modifications work in concert with replication-independent histone variants further adapted for transcriptional regulation and chromatin repair. However, little is known about how such complex regulatory pathways are orchestrated and interconnected in cells. In this work, we demonstrate the utility of mass spectrometry-based approaches to explore how different epigenetic layers interact in Arabidopsis mutants lacking certain histone chaperones. We show that defects in histone chaperone function (e.g., chromatin assembly factor-1 or nucleosome assembly protein 1 mutations) translate into an altered epigenetic landscape, which aids the plant in mitigating internal instability. We observe changes in both the levels and distribution of H2A.W.7, altogether with partial repurposing of H3.3 and changes in the key repressive (H3K27me1/2) or euchromatic marks (H3K36me1/2). These shifts in the epigenetic profile serve as a compensatory mechanism in response to impaired integration of the H3.1 histone in the fas1 mutants. Altogether, our findings suggest that maintaining genome stability involves a two-tiered approach. The first relies on flexible adjustments in histone marks, while the second level requires the assistance of chaperones for histone variant replacement.

RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer.

Wnt/ß-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce ß-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing ß-catenin turnover and propelling ligand-independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.

ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating.

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

Comparison of Yield Characteristics, Chemical Composition, Lignans Content and Antioxidant Potential of Experimentally Grown Six Linseed (Linum usitatissimum L.) Cultivars.

Linseed represents a rich source of nutritional, functional and health-beneficial compounds. Nevertheless, the chemical composition and content of bioactive compounds may be quite variable and potentially affected by various factors, including genotype and the environment. In this study, the proximate chemical composition, lignans content and antioxidant potential of six experimentally grown linseed cultivars were assessed and compared. A diagonal cultivation trial in the University of South Bohemia Experimental Station in Ceské Budejovice, Czech Republic, was established in three subsequent growing seasons (2018, 2019 and 2020). The results showed that the cultivar and growing conditions influenced most studied parameters. The lack of precipitation in May and June 2019 negatively affected the seed yield and the level of secoisolariciresinol diglucoside but did not decrease the crude protein content, which was negatively related to the oil content. The newly developed method for lignans analysis allowed the identification and quantification of secoisolariciresinol diglucoside and matairesinol. Their content correlated positively with the total polyphenol content and antioxidant assays (DPPH and ABTS radical scavenging activity), indicating the significant contribution to the biofunctional properties of linseed. On the other hand, we did not detect minor linseed lignans, pinoresinol and lariciresinol. The results of this study showed the importance of cultivar and growing conditions factors on the linseed chemical composition and the lignans content, determining its nutritional and medicinal properties.

Extraction Protocol for Parallel Analysis of Proteins and DNA from Ancient Teeth and Dental Calculus.

Dental calculus is becoming a crucial material in the study of past populations with increasing interest in its proteomic and genomic content. Here, we suggest further development of a protocol for analysis of ancient proteins and a combined approach for subsequent ancient DNA extraction. We tested the protocol on recent teeth, and the optimized protocol was applied to ancient tooth to limit the destruction of calculus as it is a precious and irreplaceable source of dietary, microbiological, and ecological information in the archeological context. Finally, the applicability of the protocol was demonstrated on samples of the ancient calculus.

Tick-borne encephalitis virus capsid protein induces translational shutoff as revealed by its structural-biological analysis.

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

Unraveling adipose tissue proteomic landscapes in severe obesity: insights into metabolic complications and potential biomarkers.

In this study, we aimed to comprehensively characterize the proteomic landscapes of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in patients with severe obesity, to establish their associations with clinical characteristics, and to identify potential serum protein biomarkers indicative of tissue-specific alterations or metabolic states. We conducted a cross-sectional analysis of 32 patients with severe obesity (16 males and 16 females) of Central European descent who underwent bariatric surgery. Clinical parameters and body composition were assessed using dual-energy X-ray absorptiometry (DXA) and bioelectrical impedance, with 15 patients diagnosed with type 2 diabetes (T2D) and 17 with hypertension. Paired SAT and VAT samples, along with serum samples, were subjected to state-of-the-art proteomics liquid chromatography-mass spectrometry (LC-MS). Our analysis identified 7,284 proteins across SAT and VAT, with 1,249 differentially expressed proteins between the tissues and 1,206 proteins identified in serum. Correlation analyses between differential protein expression and clinical traits suggest a significant role of SAT in the pathogenesis of obesity and related metabolic complications. Specifically, the SAT proteomic profile revealed marked alterations in metabolic pathways and processes contributing to tissue fibrosis and inflammation. Although we do not establish a definitive causal relationship, it appears that VAT might respond to SAT metabolic dysfunction by potentially enhancing mitochondrial activity and expanding its capacity. However, when this adaptive response is exceeded, it could possibly contribute to insulin resistance (IR) and in some cases, it may be associated with the progression to T2D. Our findings provide critical insights into the molecular foundations of SAT and VAT in obesity and may inform the development of targeted therapeutic strategies.NEW & NOTEWORTHY This study provides insights into distinct proteomic profiles of subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and serum in patients with severe obesity and their associations with clinical traits and body composition. It underscores SAT's crucial role in obesity development and related complications, such as insulin resistance (IR) and type 2 diabetes (T2D). Our findings emphasize the importance of understanding the SAT and VAT balance in energy homeostasis, proteostasis, and the potential role of SAT capacity in the development of metabolic disorders.

Toll-Like Receptor 3 Overexpression Induces Invasion of Prostate Cancer Cells, whereas Its Activation Triggers Apoptosis.

Toll-like receptor 3 (TLR3) is an endosomal receptor expressed in several immune and epithelial cells. Recent studies have highlighted its expression also in solid tumors, including prostate cancer (PCa), and have described its role primarily in the proinflammatory response and induction of apoptosis. It is up-regulated in some castration-resistant prostate cancers. However, the role of TLR3 in prostate cancer progression remains largely unknown. The current study experimentally demonstrated that exogenous TLR3 activation in PCa cell lines leads to a significant induction of secretion of the cytokines IL-6, IL-8, and interferon-ß, depending on the model and chemoresistance status. Transcriptomic analysis of TLR3-overexpressing cells revealed a functional program that is enriched for genes involved in the regulation of cell motility, migration, and tumor invasiveness. Increased motility, migration, and invasion in TLR3-overexpressing cell line were confirmed by several in vitro assays and using an orthotopic prostate xenograft model in vivo. Furthermore, TLR3-ligand induced apoptosis via cleavage of caspase-3/7 and poly (ADP-ribose) polymerase, predominantly in TLR3-overexpressing cells. These results indicate that TLR3 may be involved in prostate cancer progression and metastasis; however, it might also represent an Achilles heel of PCa, which can be exploited for targeted therapy.

Histone Variant macroH2A1.1 Enhances Nonhomologous End Joining-dependent DNA Double-strand-break Repair and Reprogramming Efficiency of Human iPSCs.

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

Trimethylacetic Anhydride-Based Derivatization Facilitates Quantification of Histone Marks at the MS1 Level.

Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom-up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples.

Benchmarking of Two Peptide Clean-Up Protocols: SP2 and Ethyl Acetate Extraction for Sodium Dodecyl Sulfate or Polyethylene Glycol Removal from Plant Samples before LC-MS/MS.

The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-µg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids.

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.

Differentiation of Sialyl Linkages Using a Combination of Alkyl Esterification and Phenylhydrazine Derivatization: Application for N-Glycan Profiling in the Sera of Patients with Lung Cancer.

Alterations in oligosaccharides and types of sialic acid (SA) attachments have been associated with different pathological states. Matrix-assisted laser desorption mass spectrometry (MS) is commonly used for glycosylation studies. However, native sialylated glycans are suppressed or not detected during MS experiments. Consequently, different approaches have been employed to neutralize the negative charge of the carboxyl group. In this study, we present the advantage of phenylhydrazine (PHN) labeling for the detection and efficient discrimination of SA linkages when this derivatization follows alkyl esterification. As expected, PHN-labeled sialylated oligosaccharides with the 2,6-linkage type can be easily recognized according to the additional shift in mass corresponding to the presence of a methyl or ethyl group. Surprisingly, oligosaccharides with the 2,3-linked SA residue instead of a lactone were detected carrying the second PHN unit. This was beneficial as no further processing after esterification was needed to stabilize the lactone form. Moreover, during tandem mass experiments, all modified glycans produced favorable fragmentation patterns with a coherent recognition of SA linkages. Although both types of esterification, herein called the EST-PHN approach, provided comparable results, methylation exhibited marginally higher linkage specificity than ethyl esterification. The simplicity and effectiveness of the methodology are demonstrated on the model compound, sialyllactose, and its applicability for biological studies is presented on N-glycan profiling in the sera of lung cancer patients.

Spruce versus Arabidopsis: different strategies of photosynthetic acclimation to light intensity change.

The acclimation of higher plants to different light intensities is associated with a reorganization of the photosynthetic apparatus. These modifications, namely, changes in the amount of peripheral antenna (LHCII) of photosystem (PS) II and changes in PSII/PSI stoichiometry, typically lead to an altered chlorophyll (Chl) a/b ratio. However, our previous studies show that in spruce, this ratio is not affected by changes in growth light intensity. The evolutionary loss of PSII antenna proteins LHCB3 and LHCB6 in the Pinaceae family is another indication that the light acclimation strategy in spruce could be different. Here we show that, unlike Arabidopsis, spruce does not modify its PSII/PSI ratio and PSII antenna size to maximize its photosynthetic performance during light acclimation. Its large PSII antenna consists of many weakly bound LHCIIs, which form effective quenching centers, even at relatively low light. This, together with sensitive photosynthetic control on the level of cytochrome b6f complex (protecting PSI), is the crucial photoprotective mechanism in spruce. High-light acclimation of spruce involves the disruption of PSII macro-organization, reduction of the amount of both PSII and PSI core complexes, synthesis of stress proteins that bind released Chls, and formation of "locked-in" quenching centers from uncoupled LHCIIs. Such response has been previously observed in the evergreen angiosperm Monstera deliciosa exposed to high light. We suggest that, in contrast to annuals, shade-tolerant evergreen land plants have their own strategy to cope with light intensity changes and the hallmark of this strategy is a stable Chl a/b ratio.

Fibroblast growth factor receptor influences primary cilium length through an interaction with intestinal cell kinase.

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

Transcriptomic and Proteomic Insights into Amborella trichopoda Male Gametophyte Functions.

Flowering plants (angiosperms) are characterized by pollen tubes (PTs; male gametophytes) carrying two immobile sperm cells that grow over long distances through the carpel toward the ovules, where double fertilization is executed. It is not understood how these reproductive structures evolved, which genes occur de novo in male gametophytes of angiosperms, and to which extent PT functions are conserved among angiosperms. To contribute to a deeper understanding of the evolution of gametophyte functions, we generated RNA sequencing data from seven reproductive and two vegetative control tissues of the basal angiosperm Amborella trichopoda and complemented these with proteomic data of pollen grains (PGs) and PTs. The eudicot model plant Arabidopsis (Arabidopsis thaliana) served as a reference organism for data analysis, as more than 200 genes have been associated with male gametophyte functions in this species. We describe methods to collect bicellular A. trichopoda PGs, to induce their germination in vitro, and to monitor PT growth and germ cell division. Transcriptomic and proteomic analyses indicate that A. trichopoda PGs are prepared for germination requiring lipids, energy, but likely also reactive oxygen species, while PTs are especially characterized by catabolic/biosynthetic and transport processes including cell wall biosynthesis and gene regulation. Notably, a number of pollen-specific genes were lacking in Arabidopsis, and the number of genes involved in pollen signaling is significantly reduced in A. trichopoda In conclusion, we provide insight into male gametophyte functions of the most basal angiosperm and establish a valuable resource for future studies on the evolution of flowering plants.

The oligomeric states of elicitins affect the hypersensitive response and resistance in tobacco.

Successful plant defence against microbial pathogens is based on early recognition and fast activation of inducible responses. Key mechanisms include detection of microbe-associated molecular patterns by membrane-localized pattern recognition receptors that induce a basal resistance response. A well-described model of such responses to pathogens involves the interactions between Solanaceae plants and proteinaceous elicitors secreted by oomycetes, called elicitins. It has been hypothesized that the formation of oligomeric structures by elicitins could be involved in their recognition and activation of defensive transduction cascades. In this study, we tested this hypothesis using several approaches, and we observed differences in tobacco plant responses induced by the elicitin ß-cryptogein (ß-CRY) and its homodimer, ß-CRYDIM. We also found that the C-terminal domain of elicitins of other ELI (true-elicitin) clades plays a significant role in stabilization of their oligomeric structure and restraint in the cell wall. In addition, covalently cross-linking ß-CRYDIM impaired the formation of signalling complexes, thereby reducing its capacity to elicit the hypersensitive response and resistance in the host plant, with no significant changes in pathogenesis-related protein expression. By revealing the details of the effects of ß-CRY dimerization on recognition and defence responses in tobacco, our results shed light on the poorly understood role of elicitins' oligomeric structures in the interactions between oomycetes and plants.

Assessing the Toxicity of 17α-Ethinylestradiol in Rainbow Trout Using a 4-Day Transcriptomics Benchmark Dose (BMD) Embryo Assay.

There is an urgent demand for more efficient and ethical approaches in ecological risk assessment. Using 17α-ethinylestradiol (EE2) as a model compound, this study established an embryo benchmark dose (BMD) assay for rainbow trout (RBT; Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs) as an alternative to live-animal tests. Embryos were exposed to graded concentrations of EE2 (measured: 0, 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects. In contrast, transcriptomics revealed responses that were in agreement with apical effects, including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L, the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the 10th percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive over-represented pathway, respectively) were within the same order of magnitude as empirically derived apical PODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving tPODs that are protective of chronic effects of EE2.

Development of a Comprehensive Toxicity Pathway Model for 17α-Ethinylestradiol in Early Life Stage Fathead Minnows (Pimephales promelas).

There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; Pimephales promelas) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.