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Pathogens are ubiquitous and a constant threat to their hosts, which has led to the evolution of sophisticated immune systems in bacteria, archaea and eukaryotes. Bacterial immune systems encode an astoundingly large array of antiviral (antiphage) systems, and recent investigations have identified unexpected similarities between the immune systems of bacteria and animals. In this Review, we discuss advances in our understanding of the bacterial innate immune system and highlight the components, strategies and pathogen restriction mechanisms conserved between bacteria and eukaryotes. We summarize evidence for the hypothesis that components of the human immune system originated in bacteria, where they first evolved to defend against phages. Further, we discuss shared mechanisms that pathogens use to overcome host immune pathways and unexpected similarities between bacterial immune systems and interbacterial antagonism. Understanding the shared evolutionary path of immune components across domains of life and the successful strategies that organisms have arrived at to restrict their pathogens will enable future development of therapeutics that activate the human immune system for the precise treatment of disease., (© 2024. Springer Nature Limited.)

Molecular identification of micro- and macroorganisms based on nuclear markers has revolutionized our understanding of their taxonomy, phylogeny and ecology. Today, research on the diversity of eukaryotes in global ecosystems heavily relies on nuclear ribosomal RNA (rRNA) markers. Here, we present the research community-curated reference database EUKARYOME for nuclear ribosomal 18S rRNA, internal transcribed spacer (ITS) and 28S rRNA markers for all eukaryotes, including metazoans (animals), protists, fungi and plants. It is particularly useful for the identification of arbuscular mycorrhizal fungi as it bridges the four commonly used molecular markers-ITS1, ITS2, 18S V4-V5 and 28S D1-D2 subregions. The key benefits of this database over other annotated reference sequence databases are that it is not restricted to certain taxonomic groups and it includes all rRNA markers. EUKARYOME also offers a number of reference long-read sequences that are derived from (meta)genomic and (meta)barcoding-a unique feature that can be used for taxonomic identification and chimera control of third-generation, long-read, high-throughput sequencing data. Taxonomic assignments of rRNA genes in the database are verified based on phylogenetic approaches. The reference datasets are available in multiple formats from the project homepage, http://www.eukaryome.org., (© The Author(s) 2024. Published by Oxford University Press.)

Unnatural amino acids (UAAs) have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins. In eukaryotes, genetic code expansion (GCE) enables the incorporation of UAAs and facilitates posttranscriptional modification (PTM), which is not feasible in prokaryotic systems. GCE is also a powerful tool for cell or animal imaging, the monitoring of protein interactions in target cells, drug development, and switch regulation. Therefore, there is keen interest in utilizing GCE in eukaryotic systems. This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.)

The move from a free-living environment to a long-term residence inside a host eukaryotic cell has profound effects on bacterial function. While endosymbioses are found in many eukaryotes, from protists to plants to animals, the bacteria that form these host-beneficial relationships are even more diverse. Endosymbiont genomes can become radically smaller than their free-living relatives, and their few remaining genes show extreme compositional biases. The details of how these reduced and divergent gene sets work, and how they interact with their host cell, remain mysterious. This Unsolved Mystery reviews how genome reduction alters endosymbiont biology and highlights a "tipping point" where the loss of the ability to build a cell envelope coincides with a marked erosion of translation-related genes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 McCutcheon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA modifications add another layer of complexity to the eukaryotic genome to regulate gene expression, playing critical roles as epigenetic marks. In eukaryotes, the study of DNA epigenetic modifications has been confined to 5mC and its derivatives for decades. However, rapid developing approaches have witnessed the expansion of DNA modification reservoirs during the past several years, including the identification of 6mA, 5gmC, 4mC, and 4acC in diverse organisms. However, whether these DNA modifications function as epigenetic marks requires careful consideration. In this review, we try to present a panorama of all the DNA epigenetic modifications in eukaryotes, emphasizing recent breakthroughs in the identification of novel DNA modifications. The characterization of their roles in transcriptional regulation as potential epigenetic marks is summarized. More importantly, the pathways for generating or eliminating these DNA modifications, as well as the proteins involved are comprehensively dissected. Furthermore, we briefly discuss the potential challenges and perspectives, which should be taken into account while investigating novel DNA modifications., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phagotrophic flagellates are the main consumers of bacteria and picophytoplankton. Despite their ecological significance in the 'microbial loop', many of their predation mechanisms remain unclear. 'Typical excavates' bear a ventral groove, where prey is captured for ingestion. The consequences of feeding through a 'semi-rigid' furrow on the prey size range have not been explored. An unidentified moving element called 'the wave' that sweeps along the bottom of the groove toward the site of phagocytosis has been observed in a few species; its function is unclear. We investigated the presence, behavior, and function of the wave in four species from the three excavate clades (Discoba, Metamonada, and Malawimonadida) and found it present in all studied cases, suggesting the potential homology of this feature across all three groups. The wave displayed a species-specific behavior and was crucial for phagocytosis. The morphology of the feeding groove had an upper-prey size limit for successful prey captures, but smaller particles were not constrained. Additionally, the ingestion efficiencies were species dependent. By jointly studying these feeding traits, we speculate on adaptations to differences in food availability to better understand their ecological functions., (© 2023 International Society of Protistologists.)

The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging., (© 2024. The Author(s).)

Life codes increase in both number and variety with biological complexity. Although our knowledge of codes is constantly expanding, the evolutionary progression of organic, neural, and cultural codes in response to selection pressure remains poorly understood. Greater clarification of the selective mechanisms is achieved by investigating how major evolutionary transitions reduce spatiotemporal and energetic constraints on transmitting heritable code to offspring. Evolution toward less constrained flows is integral to enduring flow architecture everywhere, in both engineered and natural flow systems. Beginning approximately 4 billion years ago, the most basic level for transmitting genetic material to offspring was initiated by protocell division. Evidence from ribosomes suggests that protocells transmitted comma-free or circular codes, preceding the evolution of standard genetic code. This rudimentary information flow within protocells is likely to have first emerged within the geo-energetic and geospatial constraints of hydrothermal vents. A broad-gauged hypothesis is that major evolutionary transitions overcame such constraints with tri-flow adaptations. The interconnected triple flows incorporated energy-converting, spatiotemporal, and code-based informational dynamics. Such tri-flow adaptations stacked sequence splicing code on top of protein-DNA recognition code in eukaryotes, prefiguring the transition to sexual reproduction. Sex overcame the spatiotemporal-energetic constraints of binary fission with further code stacking. Examples are tubulin code and transcription initiation code in vertebrates. In a later evolutionary transition, language reduced metabolic-spatiotemporal constraints on inheritance by stacking phonetic, phonological, and orthographic codes. In organisms that reproduce sexually, each major evolutionary transition is shown to be a tri-flow adaptation that adds new levels of code-based informational exchange. Evolving biological complexity is also shown to increase the nongenetic transmissibility of code., Competing Interests: Declaration of competing interest The author declares that there is no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author. Published by Elsevier B.V. All rights reserved.)

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina , CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Raphidocystis marginata (Siemensma, 1981), Raineriophrys echinata (Rainer, 1968), and Pterocystis fortesca (Nicholls, 1983) are heliozoan protists, have been recorded only in a few localities in Europe, and considered to be rare species. These centrohelid heliozoans have been reported for the first time in Ukrainian Polissia, and we provide their morphological descriptions with new morphometric data of exoskeleton (periplast) based on Ukrainian material. The diagnostic morphological characters are illustrated by light and scanning electron microscope photographs. Their geographical distribution in Europe and biotope preference are discussed.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) is a Bioinformatics Resource Center funded by the National Institutes of Health with additional funding from the Wellcome Trust. VEuPathDB supports >600 organisms that comprise invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Since 2004, VEuPathDB has analyzed omics data from the public domain using contemporary bioinformatic workflows, including orthology predictions via OrthoMCL, and integrated the analysis results with analysis tools, visualizations, and advanced search capabilities. The unique data mining platform coupled with >3000 pre-analyzed data sets facilitates the exploration of pertinent omics data in support of hypothesis driven research. Comparisons are easily made across data sets, data types and organisms. A Galaxy workspace offers the opportunity for the analysis of private large-scale datasets and for porting to VEuPathDB for comparisons with integrated data. The MapVEu tool provides a platform for exploration of spatially resolved data such as vector surveillance and insecticide resistance monitoring. To address the growing body of omics data and advances in laboratory techniques, VEuPathDB has added several new data types, searches and features, improved the Galaxy workspace environment, redesigned the MapVEu interface and updated the infrastructure to accommodate these changes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protists, a highly diverse group of microscopic eukaryotic organisms distinct from fungi, animals and plants, exert crucial roles within the earth's biosphere. However, the genomes of only a small fraction of known protist species have been published and made publicly accessible. To address this constraint, the Protist 10 000 Genomes Project (P10K) was initiated, implementing a specialized pipeline for single-cell genome/transcriptome assembly, decontamination and annotation of protists. The resultant P10K database (https://ngdc.cncb.ac.cn/p10k/) serves as a comprehensive platform, collating and disseminating genome sequences and annotations from diverse protist groups. Currently, the P10K database has incorporated 2959 genomes and transcriptomes, including 1101 newly sequenced datasets by P10K and 1858 publicly available datasets. Notably, it covers 45% of the protist orders, with a significant representation (53% coverage) of ciliates, featuring nearly a thousand genomes/transcriptomes. Intriguingly, analysis of the unique codon table usage among ciliates has revealed differences compared to the NCBI taxonomy system, suggesting a need to revise the codon tables used for these species. Collectively, the P10K database serves as a valuable repository of genetic resources for protist research and aims to expand its collection by incorporating more sequenced data and advanced analysis tools to benefit protist studies worldwide., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryote-interacting bacteria have developed along the evolution of an arsenal of tools to interact with potential hosts and to evade their defensive responses. Among these tools, the effector proteins are gaining a special importance due to the high diversity of molecular actions that they play in the host cell, with the final aim of taking the control over the cell. Bacteria inject these effectors into the cytosol of the host cells through distinct ways, as the type III secretion system. The study of the effectors' molecular roles inside the host cell is challenging, due in part to the lack of traceability of such proteins once they are delivered by the bacteria. Here, we describe in depth a methodology that combines the increase of the bacterial effector concentration by protein expression systems with the use of heterologous hosts to facilitate the visualization of the subcellular targeting of the effector inside the host cell by fluorescence microscopy., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cells are the fundamental unit of biological organization. Although it may be easy to think of them as little more than the simple building blocks of complex organisms such as animals, single cells are capable of behaviors of remarkable apparent sophistication. This is abundantly clear when considering the diversity of form and function among the microbial eukaryotes, the protists. How might we navigate this diversity in the search for general principles of cellular behavior? Here, we review cases in which the intensive study of protists from the perspective of cellular biophysics has driven insight into broad biological questions of morphogenesis, navigation and motility, and decision making. We argue that applying such approaches to questions of evolutionary cell biology presents rich, emerging opportunities. Integrating and expanding biophysical studies across protist diversity, exploiting the unique characteristics of each organism, will enrich our understanding of general underlying principles., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Bdelloid rotifers are part of the restricted circle of multicellular animals that can withstand a wide range of genotoxic stresses at any stage of their life cycle. In this study, bdelloid rotifer Adineta vaga is used as a model to decipher the molecular basis of their extreme tolerance. Proteomic analysis shows that a specific DNA ligase, different from those usually involved in DNA repair in eukaryotes, is strongly over-represented upon ionizing radiation. A phylogenetic analysis reveals its orthology to prokaryotic DNA ligase E, and its horizontal acquisition by bdelloid rotifers and plausibly other eukaryotes. The fungus Mortierella verticillata, having a single copy of this DNA Ligase E homolog, also exhibits an increased radiation tolerance with an over-expression of this DNA ligase E following X-ray exposure. We also provide evidence that A. vaga ligase E is a major contributor of DNA breaks ligation activity, which is a common step of all important DNA repair pathways. Consistently, its heterologous expression in human cell lines significantly improves their radio-tolerance. Overall, this study highlights the potential of horizontal gene transfers in eukaryotes, and their contribution to the adaptation to extreme conditions., (© 2023. The Author(s).)

Microbial life maintains nearly all the support systems that keep the Earth habitable, yet the diversity of this vast microbial world is greatly understudied, misrepresented, and misunderstood. Even what we do know is difficult to communicate broadly because an intuitive grasp of what these tiny organisms are like is abstract, and we lack tools that would help to describe them. In this Essay, we present a series of openly available technical diagrams that illustrate the diverse range of complex body plans of microbial eukaryotes (or "protists"), as well as an illustrated tree to show the vast diversity they encompass and how they are related to the more familiar macroscopic animals, fungi, and plants. These sorts of tools are desperately needed for teaching and communication about the microbial world, which is a pressingly important problem where much improvement is needed., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Keeling, Eglit. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Host-microbe interactions are ubiquitous and play important roles in host biology, ecology, and evolution. Yet, host-microbe research has focused on inland species, whereas marine hosts and their associated microbes remain largely unexplored, especially in developing countries in the Southern Hemisphere. Here, we review the current knowledge of marine host microbiomes in the Southern Hemisphere. Our results revealed important biases in marine host species sampling for studies conducted in the Southern Hemisphere, where sponges and marine mammals have received the greatest attention. Sponge-associated microbes vary greatly across geographic regions and species. Nevertheless, besides taxonomic heterogeneity, sponge microbiomes have functional consistency, whereas geography and aging are important drivers of marine mammal microbiomes. Seabird and macroalgal microbiomes in the Southern Hemisphere were also common. Most seabird microbiome has focused on feces, whereas macroalgal microbiome has focused on the epibiotic community. Important drivers of seabird fecal microbiome are aging, sex, and species-specific factors. In contrast, host-derived deterministic factors drive the macroalgal epibiotic microbiome, in a process known as "microbial gardening". In turn, marine invertebrates (especially crustaceans) and fish microbiomes have received less attention in the Southern Hemisphere. In general, the predominant approach to study host marine microbiomes has been the sequencing of the 16S rRNA gene. Interestingly, there are some marine holobiont studies ( i.e ., studies that simultaneously analyze host ( e.g ., genomics, transcriptomics) and microbiome ( e.g ., 16S rRNA gene, metagenome) traits), but only in some marine invertebrates and macroalgae from Africa and Australia. Finally, we introduce an ongoing project on the surface microbiome of key species in the Strait of Magellan. This is an international project that will provide novel microbiome information of several species in the Strait of Magellan. In the short-term, the project will improve our knowledge about microbial diversity in the region, while long-term potential benefits include the use of these data to assess host-microbial responses to the Anthropocene derived climate change., Competing Interests: Lía Ramírez-Fernández is employed by Centro de Desarrollo de Biotecnología Industrial y Bioproducto. E. Paola Acuña-Gómez & Valeria Souza are employed by Centro de Estudios del Cuaternario de Fuego. Manuel Ochoa-Sánchez is a doctoral student affiliated to Centro de Estudios del Cuaternario de Fuego. Luis E. Eguiarte & Valeria Souza are Academic Editors for PeerJ., (© 2023 Ochoa-Sánchez et al.)

In a recent article, we presented evidence demonstrating the existence of hidden y-stories within the genomes of humans and canines. These stories were found not only in the non-protein-coding regions but also within the genetic regions and the sequence of exons. Consequently, we are now exploring whether these discoveries are unique to humans and dogs or if they are more widely distributed throughout the cellular world. To approach this question, we embarked on an investigation of the genomes of various species across Whittaker's five kingdoms, namely Animalia, Plantae, Fungi, Protista, and Monera. Through online resources, we obtained and analysed whole-genome sequences of one avian species, one fish species, one reptile species, and one invertebrate species within the Animalia kingdom. Furthermore, we examined the genomes of one plant species, one fungus species, one protozoan species, and two bacterial species. Employing the same methods as in our prior studies, our findings in this study align with our proto knowledge hypothesis, suggesting that all living cells possess a repository of hidden y-information which determines the cellular design, sustains its overall functionality, and governs its performance and behaviour throughout its lifespan until death. We briefly explain life as a bio-linguistic phenomenon and future projects.

Plant virual infections are mainly caused by plant-virus parasitism which affects ecological communities. Some viruses are highly pathogen specific that can infect only specific plants, while some can cause widespread harm, such as tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). After a virus infects the host, undergoes a series of harmful effects, including the destruction of host cell membrane receptors, changes in cell membrane components, cell fusion, and the production of neoantigens on the cell surface. Therefore, competition between the host and the virus arises. The virus starts gaining control of critical cellular functions of the host cells and ultimately affects the fate of the targeted host plants. Among these critical cellular processes, alternative splicing (AS) is an essential posttranscriptional regulation process in RNA maturation, which amplify host protein diversity and manipulates transcript abundance in response to plant pathogens. AS is widespread in nearly all human genes and critical in regulating animal-virus interactions. In particular, an animal virus can hijack the host splicing machinery to re-organize its compartments for propagation. Changes in AS are known to cause human disease, and various AS events have been reported to regulate tissue specificity, development, tumour proliferation, and multi-functionality. However, the mechanisms underlying plant-virus interactions are poorly understood. Here, we summarize the current understanding of how viruses interact with their plant hosts compared with humans, analyze currently used and putative candidate agrochemicals to treat plant-viral infections, and finally discussed the potential research hotspots in the future. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing., (© 2023 Wiley Periodicals LLC.)

Signal perception is a key function in regulating biological activities and adapting to changing environments. Per-Arnt-Sim (PAS) domains are ubiquitous sensors found in diverse receptors in bacteria, archaea, and eukaryotes, but their origins, distribution across the tree of life, and extent of their functional diversity are not fully characterized. Here, we show that using sequence conservation and structural information, it is possible to propose specific and potential functions for a large portion of nearly 3 million PAS domains. Our analysis suggests that PAS domains originated in bacteria and were horizontally transferred to archaea and eukaryotes. We reveal that gas sensing via a heme cofactor evolved independently in several lineages, whereas redox and light sensing via flavin adenine dinucleotide and flavin mononucleotide cofactors have the same origin. The close relatedness of human PAS domains to those in bacteria provides an opportunity for drug design by exploring potential natural ligands and cofactors for bacterial homologs.

The aphelids, intracellular parasitoids of algae, represent a large cluster of species sister to Fungi in molecular phylogenetic trees. Sharing a common ancestor with Fungi, they are very important in terms of evolution of these groups of Holomycota. Aphelid life cycle being superficially similar to that of Chytridiomycetes is understudied. We have found in the aphelids a new stage-big multiflagellar and amoeboid cells, formed from a plasmodium that has two sorts of nuclei after trophic stage fusion. The families of protein-coding genes involved in the vegetative cell fusion in Opisthokonta were also discussed., (© 2023 International Society of Protistologists.)

Background: The Earth Biogenome Project has rapidly increased the number of available eukaryotic genomes, but most released genomes continue to lack annotation of protein-coding genes. In addition, no transcriptome data is available for some genomes., Results: Various gene annotation tools have been developed but each has its limitations. Here, we introduce GALBA, a fully automated pipeline that utilizes miniprot, a rapid protein-to-genome aligner, in combination with AUGUSTUS to predict genes with high accuracy. Accuracy results indicate that GALBA is particularly strong in the annotation of large vertebrate genomes. We also present use cases in insects, vertebrates, and a land plant. GALBA is fully open source and available as a docker image for easy execution with Singularity in high-performance computing environments., Conclusions: Our pipeline addresses the critical need for accurate gene annotation in newly sequenced genomes, and we believe that GALBA will greatly facilitate genome annotation for diverse organisms., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Cilia are fundamental organelles of eukaryotes with diverse functions spanning from cell motility to sensory perception. A new study presenting genomes from parasitic horsehair worms reveals highly reduced genomes that have lost the molecular machinery needed to make cilia., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Selenium (Se) is an important microelement for animal health. However, the knowledge about the effects of Se supplementation on rumen eukaryotic community remains less explored. In this study, the ruminal eukaryotic diversity in three months old Shaanbei white cashmere wether goats, with body weight (26.18 ± 2.71) kg, fed a basal diet [0.016 mg/kg Se dry matter (DM), control group (CG)] were compared to those animals given basal diet supplemented with different levels of organic Se in the form of Selenohomolanthionine (SeHLan), namely low Se group (LSE, 0.3 mg/kg DM), medium Se group (MSE, 0.6 mg/kg Se DM) and high Se group (HSE, 1.2 mg/kg DM) using 18S rRNA amplicon sequencing. Illumina sequencing generated 2,623,541 reads corresponding to 3123 operational taxonomic units (OTUs). Taxonomic analysis revealed that Eukaryota (77.95%) and Fungi (14.10%) were the dominant eukaryotic kingdom in all samples. The predominant rumen eukaryotic phylum was found to be Ciliophora (92.14%), while fungal phyla were dominated by Ascomycota (40.77%), Basidiomycota (23.77%), Mucoromycota (18.32%) and unidentified&#95;Fungi (13.89%). The dominant eukaryotic genera were found to be Entodinium (55.44%), Ophryoscolex (10.51%) and Polyplastron (10.19%), while the fungal genera were dominanted by Mucor (15.39%), Pichia (9.88%), Aspergillu (8.24%), Malassezia (7.73%) and unidentified&#95;Neocallimastigaceae (7.72%). The relative abundance of eukaryotic genera Ophryoscolex, Enoploplastron and fungal genus Mucor were found to differ significantly among the four treatment groups (P < 0.05). Moreover, Spearman correlation analysis revealed that the ciliate protozoa and fungi were negatively correlated with each other. The results of this study provided newer information about the effects of Se on rumen eukaryotic diversity patterns using 18s rRNA high-throughput sequencing technology., (© 2023. Springer Nature Limited.)

Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analyses. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from https://github.com/cmbi/compact-bio., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 van Strien et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To study unknown proteins on a large scale, a reference system has been set up for the three better studied eukaryotic kingdoms, built with 36 proteomes as taxonomically diverse as possible. Proteins from 362 other eukaryotic proteomes with no known homologue in this set were then analyzed, focusing noteworthy on singletons, that is, on such proteins with no known homologue in their own proteome. Consistently, for a given species, no more than 12% of the singletons thus found are known at the protein level, according to Uniprot. In addition, since they rely on the information found in the alignment of homologous sequences, predictions of AlphaFold2 for their tridimensional structure are poor. In the case of metazoan species, the number of singletons rarely exceeds 1000 for the species the closest to the reference system (divergence times below 75 Myr). Interestingly, in the cases of viridiplantae and fungi, larger amounts of singletons are found for such species, as if the timescale on which singletons are added to proteomes were different in metazoa and in other eukaryotic kingdoms. In order to confirm this phenomenon, further studies of proteomes closer to those of the reference system are, however, needed., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A large part of the soil protist diversity is missed in metabarcoding studies based on 0.25 g of soil environmental DNA (eDNA) and universal primers due to ca. 80% co-amplification of non-target plants, animals and fungi. To overcome this problem, enrichment of the substrate used for eDNA extraction is an easily implemented option but its effect has not yet been tested. In this study, we evaluated the effect of a 150 μm mesh size filtration and sedimentation method to improve the recovery of protist eDNA, while reducing the co-extraction of plant, animal and fungal eDNA, using a set of contrasted forest and alpine soils from La Réunion, Japan, Spain and Switzerland. Total eukaryotic diversity was estimated by V4 18S rRNA metabarcoding and classical amplicon sequence variant calling. A 2- to 3-fold enrichment in shelled protists (Euglyphida, Arcellinida and Chrysophyceae) was observed at the sample level with the proposed method, with, at the same time, a 2-fold depletion of Fungi and a 3-fold depletion of Embryophyceae. Protist alpha diversity was slightly lower in filtered samples due to reduced coverage in Variosea and Sarcomonadea, but significant differences were observed in only one region. Beta diversity varied mostly between regions and habitats, which explained the same proportion of variance in bulk soil and filtered samples. The increased resolution in soil protist diversity estimates provided by the filtration-sedimentation method is a strong argument in favour of including it in the standard protocol for soil protist eDNA metabarcoding studies., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Despite differences in their overall metabolism, eukaryotes share a common mitochondrial biochemistry. We investigated how this fundamental biochemistry supports overall metabolism using a high-resolution carbon isotope approach, position-specific isotope analysis. We measured carbon isotope 13 C/ 12 C cycling in animals, focusing on amino acids that are formed in mitochondrial reactions and are most metabolically active. Carboxyl isotope determinations for amino acids showed strong signals related to common biochemical pathways. Contrasting isotope patterns were measured for metabolism associated with major life history patterns, including growth and reproduction. Turnover of proteins and lipids as well as gluoconeogensis dynamics could be estimated for these metabolic life histories. The high-resolution isotomics measurements fingerprinted metabolism and metabolic strategies across the eukaryotic animal kingdom, yielding results for humans, ungulates, whales, and diverse fish and invertebrates in a nearshore marine food web.

Many cell biological facts that can be found in dedicated scientific textbooks are based on findings originally made in humans and/or other mammals, including respective tissue culture systems. They are often presented as if they were universally valid, neglecting that many aspects differ-in part considerably-between the three major kingdoms of multicellular eukaryotic life, comprising animals, plants and fungi. Here, we provide a comparative cross-kingdom view on the basic cell biology across these lineages, highlighting in particular essential differences in cellular structures and processes between phyla. We focus on key dissimilarities in cellular organization, e.g. regarding cell size and shape, the composition of the extracellular matrix, the types of cell-cell junctions, the presence of specific membrane-bound organelles and the organization of the cytoskeleton. We further highlight essential disparities in important cellular processes such as signal transduction, intracellular transport, cell cycle regulation, apoptosis and cytokinesis. Our comprehensive cross-kingdom comparison emphasizes overlaps but also marked differences between the major lineages of the three kingdoms and, thus, adds to a more holistic view of multicellular eukaryotic cell biology., (© 2023. The Author(s).)

Protozoa are eukaryotic organisms that play a crucial role in nutrient cycling and maintaining balance in the food web. Predation, symbiosis and parasitism are three types of interactions between protozoa and bacteria. However, not all bacterial species are equally susceptible to protozoan predation as many are capable of defending against predation in numerous ways and may even establish either a symbiotic or parasitic life-style. Biofilm formation is one such mechanism by which bacteria can survive predation. Structural and chemical components of biofilms enhance resistance to predation compared to their planktonic counterparts. Predation on biofilms gives rise to phenotypic and genetic heterogeneity in prey that leads to trade-offs in virulence in other eukaryotes. Recent advances, using molecular and genomics techniques, allow us to generate new information about the interactions of protozoa and biofilms of prey bacteria. This review presents the current state of the field on impacts of protozoan predation on biofilms. We provide an overview of newly gathered insights into (i) molecular mechanisms of predation resistance in biofilms, (ii) phenotypic and genetic diversification of prey bacteria, and (iii) evolution of virulence as a consequence of protozoan predation on biofilms., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

DNA methylation constitutes one of the pillars of epigenetics, relying on covalent bonds for addition and/or removal of chemically distinct marks within the major groove of the double helix. DNA methyltransferases, enzymes which introduce methyl marks, initially evolved in prokaryotes as components of restriction-modification systems protecting host genomes from bacteriophages and other invading foreign DNA. In early eukaryotic evolution, DNA methyltransferases were horizontally transferred from bacteria into eukaryotes several times and independently co-opted into epigenetic regulatory systems, primarily via establishing connections with the chromatin environment. While C5-methylcytosine is the cornerstone of plant and animal epigenetics and has been investigated in much detail, the epigenetic role of other methylated bases is less clear. The recent addition of N4-methylcytosine of bacterial origin as a metazoan DNA modification highlights the prerequisites for foreign gene co-option into the host regulatory networks, and challenges the existing paradigms concerning the origin and evolution of eukaryotic regulatory systems., (© 2023 Wiley Periodicals LLC.)

Metal contamination is a global issue impacting biodiversity in urbanised estuaries. Traditional methods to assess biodiversity are time consuming, costly and often exclude small or cryptic organisms due to difficulties with morphological identification. Metabarcoding approaches have been increasingly recognised for their utility in monitoring, however studies have focused on freshwater and marine systems despite the ecological significance of estuaries. We targeted estuarine eukaryote communities within the sediments of Australia's largest urbanised estuary, where a history of industrial activity has resulted in a metal contamination gradient. We identified specific eukaryote families with significant correlations with bioavailable metal concentrations, indicating sensitivity or tolerance to specific metals. While polychaete families Terebellidae and Syllidae demonstrated tolerance to the contamination gradient, members of the meio- and microfaunal communities including diatoms, dinoflagellates and nematodes displayed sensitivities. These may have high value as indicators but are frequently missed in traditional surveys due to sampling limitations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Phylogenetic reconstruction using concatenated loci ("phylogenomics" or "supermatrix phylogeny") is a powerful tool for solving evolutionary splits that are poorly resolved in single gene/protein trees. However, recent phylogenomic attempts to resolve the eukaryote root have yielded conflicting results, along with claims of various artifacts hidden in the data. We have investigated these conflicts using two new methods for assessing phylogenetic conflict. ConJak uses whole marker (gene or protein) jackknifing to assess deviation from a central mean for each individual sequence, whereas ConWin uses a sliding window to screen for incongruent protein fragments (mosaics). Both methods allow selective masking of individual sequences or sequence fragments in order to minimize missing data, an important consideration for resolving deep splits with limited data. Analyses focused on a set of 76 eukaryotic proteins of bacterial ancestry previously used in various combinations to assess the branching order among the three major divisions of eukaryotes: Amorphea (mainly animals, fungi, and Amoebozoa), Diaphoretickes (most other well-known eukaryotes and nearly all algae) and Excavata, represented here by Discoba (Jakobida, Heterolobosea, and Euglenozoa). ConJak analyses found strong outliers to be concentrated in undersampled lineages, whereas ConWin analyses of Discoba, the most undersampled of the major lineages, detected potentially incongruent fragments scattered throughout. Phylogenetic analyses of the full data using an LG-gamma model support a Discoba sister scenario (neozoan-excavate root), which rises to 99-100% bootstrap support with data masked according to either protocol. However, analyses with two site-specific (CAT) mixture models yielded widely inconsistent results and a striking sensitivity to missing data. The neozoan-excavate root places Amorphea and Diaphoretickes as more closely related to each other than either is to Discoba, a fundamental relationship that should remain unaffected by additional taxa. [CAT-GTR; Discoba; eukaryote tree of life; HGT; jackknife; mixture models; mosaic genes; phylogenomics; sliding window; supermatrix.]., (© The Author(s) 2022. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Regulated cellular aggregation is an essential process for development and healing in many animal tissues. In some animals and a few distantly related unicellular species, cellular aggregation is regulated by diffusible chemical cues. However, it is unclear whether regulated cellular aggregation was part of the life cycles of the first multicellular animals and/or their unicellular ancestors. To fill this gap, we investigated the triggers of cellular aggregation in one of animals' closest unicellular living relatives-the filasterean Capsaspora owczarzaki . We discovered that Capsaspora aggregation is induced by chemical cues, as observed in some of the earliest branching animals and other unicellular species. Specifically, we found that calcium ions and lipids present in lipoproteins function together to induce aggregation of viable Capsaspora cells. We also found that this multicellular stage is reversible as depletion of the cues triggers disaggregation, which can be overcome upon reinduction. Our finding demonstrates that chemically regulated aggregation is important across diverse members of the holozoan clade. Therefore, this phenotype was plausibly integral to the life cycles of the unicellular ancestors of animals.

The latest omicron variants are emerging with mutations in the receptor binding domain (RBD) that confer immune evasion and resistance against current vaccines. Such variants have raised the peril of future vaccine effectiveness, as leading vaccines target the spike protein. Type-IV hypersensitivity, and other ailments due to the dominant Th1 response by leading vaccines, is also to be resolved. Therefore, vaccine that target diverse SARS-CoV-2 proteins and provide broad-spectrum protection and a balanced Th1 and Th2 response is an indispensable armament against the pandemic. In that prospect, a novel dual expression plasmid pJHL270 was developed and demonstrated the expression of omicron antigens exogenously from Salmonella and endogenously in the host cells. The simultaneous activation of MHC class I and II molecules culminated in a balanced Th1 and Th2 response, which was evident through the upsurge of IgG, IgA antibodies, IgG2a/IgG1 ratio, cytokine responses and CD4+, CD8+ T-lymphocytes. The level of CD44+ cells showed the trigger for Th1 and Th2 balance and memory-cell activation for long-lasting immunity. The level of IFN-γ + cells and neutralizing antibodies signifies the anti-viral response. The vaccine protected the hamsters from BA.5 and BA.2.75 omicron viral-challenge, exhibited a significant reduction in lung viral-load and histopathological lesions. In addition to two-way antigen expression and bilateral immune elicitation, this Salmonella-based vaccine delivery system can be prospectively applied to humans and a broad range of animals as a convenient alternative to viral and chemical vaccine delivery approaches., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The ichthyosporean Sphaeroforma arctica, a protist closely related to animals, displays coenocytic development followed by cellularization and cell release. A new study reveals that the nuclear-to-cytoplasmic ratio drives cellularization in these fascinating organisms., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The ratio of nuclear content to cytoplasmic volume (N/C ratio) is a key regulator driving the maternal-to-zygotic transition in most animal embryos. Altering this ratio often impacts zygotic genome activation and deregulates the timing and outcome of embryogenesis. 1 Despite being ubiquitous across animals, little is known about when the N/C ratio evolved to control multicellular development. Such capacity either originated with the emergence of animal multicellularity or was co-opted from the mechanisms present in unicellular organisms. , An effective strategy to tackle this question is to investigate the close relatives of animals exhibiting life cycles with transient multicellular stages. 2 , 3 Despite being ubiquitous across animals, little is known about when the N/C ratio evolved to control multicellular development. Such capacity either originated with the emergence of animal multicellularity or was co-opted from the mechanisms present in unicellular organisms. 4 An effective strategy to tackle this question is to investigate the close relatives of animals exhibiting life cycles with transient multicellular stages. 5 Among these are ichthyosporeans, a lineage of protists undergoing coenocytic development followed by cellularization and cell release. 6 , 7 , 8 During cellularization, a transient multicellular stage resembling animal epithelia is generated, offering a unique opportunity to examine whether the N/C ratio regulates multicellular development. Here, we use time-lapse microscopy to characterize how the N/C ratio affects the life cycle of the best-studied ichthyosporean model, Sphaeroforma arctica. We uncover that the last stages of cellularization coincide with a significant increase in the N/C ratio. Increasing the N/C ratio by reducing the coenocytic volume accelerates cellularization, whereas decreasing the N/C ratio by lowering the nuclear content halts it. Moreover, centrifugation and pharmacological inhibitor experiments suggest that the N/C ratio is locally sensed at the cortex and relies on phosphatase activity. Altogether, our results show that the N/C ratio drives cellularization in S. arctica, suggesting that its capacity to control multicellular development predates animal emergence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Eukaryotic genomes contain a variety of endogenous viral elements (EVEs), which are mostly derived from RNA and ssDNA viruses that are no longer functional and are considered to be "genomic fossils." Genomic surveys of EVEs, however, are strongly biased toward animals and plants, whereas protists, which represent the majority of eukaryotic diversity, remain poorly represented. Here, we show that protist genomes harbor tens to thousands of diverse, ~14 to 40 kbp long dsDNA viruses. These EVEs, composed of virophages, Polinton-like viruses, and related entities, have remained hitherto hidden owing to poor sequence conservation between virus groups and their repetitive nature that precluded accurate short-read assembly. We show that long-read sequencing technology is ideal for resolving virus insertions. Many protist EVEs appear intact, and most encode integrases, which suggests that they have actively colonized hosts across the tree of eukaryotes. We also found evidence for gene expression in host transcriptomes and that closely related virophage and Polinton-like virus genomes are abundant in viral metagenomes, indicating that many EVEs are probably functional viruses.

Sterane molecular fossils are broadly interpreted as eukaryotic biomarkers, although diverse bacteria also produce sterols. Steranes with side-chain methylations can act as more specific biomarkers if their sterol precursors are limited to particular eukaryotes and are absent in bacteria. One such sterane, 24-isopropylcholestane, has been attributed to demosponges and potentially represents the earliest evidence for animals on Earth, but enzymes that methylate sterols to give the 24-isopropyl side-chain remain undiscovered. Here, we show that sterol methyltransferases from both sponges and yet-uncultured bacteria function in vitro and identify three methyltransferases from symbiotic bacteria each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We demonstrate that bacteria have the genomic capacity to synthesize side-chain alkylated sterols, and that bacterial symbionts may contribute to 24-isopropyl sterol biosynthesis in demosponges. Together, our results suggest bacteria should not be dismissed as potential contributing sources of side-chain alkylated sterane biomarkers in the rock record., (© 2023. The Author(s).)

Almost all eukaryotes undergo sexual reproduction to generate diversity and select for fitness in their population pools. Interestingly, the systems by which sex is defined are highly diverse and can even differ between evolutionarily closely related species. While the most commonly known form of sex determination involves males and females in animals, eukaryotic microbes can have as many as thousands of different mating types for the same species. Furthermore, some species have found alternatives to sexual reproduction and prefer to grow clonally and yet undergo infrequent facultative sexual reproduction. These organisms are mainly invertebrates and microbes, but several examples are also present among vertebrates suggesting that alternative modes of sexual reproduction evolved multiple times throughout evolution. In this review, we summarize the sex-determination modes and variants of sexual reproduction found across the eukaryotic tree of life and suggest that eukaryotic microbes provide unique opportunities to study these processes in detail. We propose that understanding variations in modes of sexual reproduction can serve as a foundation to study the evolution of sex and why and how it evolved in the first place.

Ancestral sequence reconstruction is a fundamental aspect of molecular evolution studies and can trace small-scale sequence modifications through the evolution of genomes and species. In contrast, fine-grained reconstructions of ancestral genome organizations are still in their infancy, limiting our ability to draw comprehensive views of genome and karyotype evolution. Here we reconstruct the detailed gene contents and organizations of 624 ancestral vertebrate, plant, fungi, metazoan and protist genomes, 183 of which are near-complete chromosomal gene order reconstructions. Reconstructed ancestral genomes are similar to their descendants in terms of gene content as expected and agree precisely with reference cytogenetic and in silico reconstructions when available. By comparing successive ancestral genomes along the phylogenetic tree, we estimate the intra- and interchromosomal rearrangement history of all major vertebrate clades at high resolution. This freely available resource introduces the possibility to follow evolutionary processes at genomic scales in chronological order, across multiple clades and without relying on a single extant species as reference., (© 2023. The Author(s).)

The tricarboxylic acid cycle is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive cross-linking mass spectrometry data supported by phylogenetic analyses, we provide evidence that MRPS36 is an important member of the eukaryotic OGDHC, with no prokaryotic orthologues. Comparative sequence analysis and computational structure predictions reveal that, in contrast with bacteria and archaea, eukaryotic E2o does not contain the peripheral subunit-binding domain (PSBD), for which we propose that MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC of approximately 3.45 MDa with novel mechanistic insights.

Low-level DNA N 6 -methyldeoxyadenosine (DNA-m6A) has recently been reported across various eukaryotes. Although anti-m6A antibody-based approaches are commonly used to measure DNA-m6A levels, this approach is known to be confounded by DNA secondary structures, RNA contamination, and bacterial contamination. To evaluate for these confounding features, we introduce an approach for systematically validating the selectivity of antibody-based DNA-m6A methods and use a highly selective anti-DNA-m6A antibody to reexamine patterns of DNA-m6A in C. reinhardtii , A. thaliana , and D. melanogaster Our findings raise caution about the use of antibody-based methods for endogenous m6A quantification and mapping in eukaryotes., (© 2023 Debo et al.; Published by Cold Spring Harbor Laboratory Press.)

The T7 system is an orthogonal transcription-system, which is characterized by simplicity, higher efficiency, and higher processivity, and it is used for protein or mRNA synthesis in various biological-systems. In comparison with prokaryotes, the construction of the T7 expression system is still on-going in eukaryotes, but it shows greatly applicable prospects. In the present paper, development of T7 expression system construction in eukaryotes is reviewed, including its construction in animal (mammalian cells, trypanosomatid protozoa, Xenopus oocytes, zebrafish), plant, and microorganism and its application in vaccine production and gene therapy. In addition, the innate challenges of T7 expression system construction in eukaryote and its potential application in vaccine production and gene therapy are discussed., (© 2022 Wiley-VCH GmbH.)

The ubiquitin-26S proteasome system and autophagy are two major protein degradation machineries encoded in all eukaryotic organisms. While the UPS is responsible for the turnover of short-lived and/or soluble misfolded proteins under normal growth conditions, the autophagy-lysosomal/vacuolar protein degradation machinery is activated under stress conditions to remove long-lived proteins in the forms of aggregates, either soluble or insoluble, in the cytoplasm and damaged organelles. Recent discoveries suggested an integrative function of these two seemly independent systems for maintaining the proteome homeostasis. One such integration is represented by their reciprocal degradation, in which the small 76-amino acid peptide, ubiquitin, plays an important role as the central signaling hub. In this review, we summarized the current knowledge about the activity control of proteasome and autophagosome at their structural organization, biophysical states, and turnover levels from yeast and mammals to plants. Through comprehensive literature studies, we presented puzzling questions that are awaiting to be solved and proposed exciting new research directions that may shed light on the molecular mechanisms underlying the biological function of protein degradation.

Most prokaryotic proteins consist of a single structural domain (SD) with little intrinsically disordered regions (IDRs) that by themselves do not adopt stable structures, whereas the typical eukaryotic protein comprises multiple SDs and IDRs. How eukaryotic proteins evolved to differ from prokaryotic proteins has not been fully elucidated. Here, we found that the longer the internal exons are, the more frequently they encode IDRs in eight eukaryotes including vertebrates, invertebrates, a fungus, and plants. Based on this observation, we propose the "small bang" model from the proteomic viewpoint: the protoeukaryotic genes had no introns and mostly encoded one SD each, but a majority of them were subsequently divided into multiple exons (step 1). Many exons unconstrained by SDs elongated to encode IDRs (step 2). The elongated exons encoding IDRs frequently facilitated the acquisition of multiple SDs to make the last common ancestor of eukaryotes (step 3). One prediction of the model is that long internal exons are mostly unconstrained exons. Analytical results of the eight eukaryotes are consistent with this prediction. In support of the model, we identified cases of internal exons that elongated after the rat-mouse divergence and discovered that the expanded sections are mostly in unconstrained exons and preferentially encode IDRs. The model also predicts that SDs followed by long internal exons tend to have other SDs downstream. This prediction was also verified in all the eukaryotic species analyzed. Our model accounts for the dichotomy between prokaryotic and eukaryotic proteins and proposes a selective advantage conferred by IDRs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

This article provides an updated review on the evolution of mitochondria from bacteria, which were likely related to extant alphaproteobacteria. Particular attention is given to the timeline of oxygen history on Earth and the entwined phases of eukaryotic evolution that produced the animals that still populate our planet. Mitochondria of early-branching unicellular eukaryotes and plants appear to retain partial or vestigial traits that were directly inherited from the alphaproteobacterial ancestors of the organelles. Most of such traits define the current aerobic physiology of mitochondria. Conversely, the anaerobic traits that would be essential in the syntrophic associations postulated for the evolution of eukaryotic cells are scantly present in extant alphaproteobacteria, and therefore cannot help defining from which bacterial lineage the ancestors of mitochondria originated. This question has recently been addressed quantitatively, reaching the novel conclusion that marine bacteria related to Iodidimonas may be the living relatives of protomitochondria. Additional evidence is presented that either support or does not contrast this novel view of the bacterial origin of mitochondria., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Procedures are described for the heterologous expression and purification of the mitochondrial-bound enzymes human and rat monoamine oxidases A and B and zebrafish MAO in the yeast Pichia pastoris. Enzyme expression is under control of a methanol oxidase promoter and similar procedures have been developed for the preparation of membrane particles and detergent solubilization of the functional enzymes. Similarities and differences are described in the procedures for purification of the respective enzymes using standard column chromatographic techniques to provide enzyme yields in the range of 100-300 mg from 1 L of cell culture., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Transgenic approaches using I-SceI are powerful genome modification methods for creating heritable modifications in eukaryotic genomes. Such modifications are ideal for studying putative promoters and their temporal and spatial expression patterns in real time, in vivo. Central to this process is the initial engineering of a plasmid construct containing multiple DNA modules in a specific order prior to the integration into the target genome. One popular way of doing this is based upon the pGateway system, the modular form of which described in this chapter is known as pTransgenesis. We will initially describe the protocol of obtaining the plasmid construct containing the required sequence modules, and then the process of integrating the construct into the genome of a Xenopus embryo via co-injection with I-SceI and subsequent screening for transgenics., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)