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Reuterin (3-hyrdoxypropionaldehyde (3-HPA)) is a highly potent metabolite of L. reuteri, which has applications in food, health, and veterinary sectors. Similar to other natural antimicrobial compounds, the approval of reuterin as a bio-preservative or therapeutic agent by regulatory agencies relies on sufficient data on its cytotoxicity and behavior in the gastrointestinal environment. Although the antimicrobial activity of reuterin has been broadly studied, its safety and toxicity are yet to be explored in detail. In this study, the stability and activity of reuterin were investigated in the gastrointestinal tract using in vitro models simulating gastrointestinal conditions. In addition, hemolytic activity and in vitro cytotoxicity of reuterin were evaluated by neutral red assay and lactate dehydrogenase (LDH) colorimetric assay using the same cell line. Activity of reuterin was observed to be stable during gastrointestinal transit. Viability and membrane integrity of cells remained unaltered by reuterin up to 1080 mM concentration. Furthermore, no hemolysis was observed in blood cells exposed to 270 mM reuterin. This study provides unique and highly relevant in vitro data regarding gastrointestinal behavior and toxicity of reuterin. In conclusion, the current study indicates that within a certain concentration range, reuterin can be safely used in bio-preservation and therapeutics applications. However, further in vivo studies are required to confirm these findings.

All organisms, fundamentally, are made from the same raw material, namely the elements of the periodic table. Biochemical diversity is achieved by how these elements are utilized, for what purpose, and in which physical location. Determining elemental distributions, especially those of trace elements that facilitate metabolism as cofactors in the active centers of essential enzymes, can determine the state of metabolism, the nutritional status, or the developmental stage of an organism. Photosynthetic eukaryotes, especially algae, are excellent subjects for quantitative analysis of elemental distribution. These microbes utilize unique metabolic pathways that require various trace nutrients at their core to enable their operation. Photosynthetic microbes also have important environmental roles as primary producers in habitats with limited nutrient supplies or toxin contaminations. Accordingly, photosynthetic eukaryotes are of great interest for biotechnological exploitation, carbon sequestration, and bioremediation, with many of the applications involving various trace elements and consequently affecting their quota and intracellular distribution. A number of diverse applications were developed for elemental imaging, allowing subcellular resolution, with X-ray fluorescence microscopy (XFM, XRF) being at the forefront, enabling quantitative descriptions of intact cells in a non-destructive method. This Tutorial Review summarizes the workflow of a quantitative, single-cell elemental distribution analysis of a eukaryotic alga using XFM.

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2-4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

There is massive variation in intron numbers across eukaryotic genomes, yet the major drivers of intron content during evolution remain elusive. Rapid intron loss and gain in some lineages contrast with long-term evolutionary stasis in others. Episodic intron gain could be explained by recently discovered specialized transposons called Introners, but so far Introners are only known from a handful of species. Here, we performed a systematic search across 3,325 eukaryotic genomes and identified 27,563 Introner-derived introns in 175 genomes (5.2%). Species with Introners span remarkable phylogenetic diversity, from animals to basal protists, representing lineages whose last common ancestor dates to over 1.7 billion years ago. Aquatic organisms were 6.5 times more likely to contain Introners than terrestrial organisms. Introners exhibit mechanistic diversity but most are consistent with DNA transposition, indicating that Introners have evolved convergently hundreds of times from nonautonomous transposable elements. Transposable elements and aquatic taxa are associated with high rates of horizontal gene transfer, suggesting that this combination of factors may explain the punctuated and biased diversity of species containing Introners. More generally, our data suggest that Introners may explain the episodic nature of intron gain across the eukaryotic tree of life. These results illuminate the major source of ongoing intron creation in eukaryotic genomes.

Subcellular organization is essential for life. Cells organize their functions into organelles to concentrate their machinery and supplies for optimal efficiency. Likewise, viruses organize their replication machinery into compartments or factories within their host cells for optimal replicative efficiency. In this review, we discuss how DNA viruses that infect both eukaryotic cells and bacteria assemble replication compartments for synthesis of progeny viral DNA and transcription of the viral genome. Eukaryotic DNA viruses assemble replication compartments in the nucleus of the host cell while DNA bacteriophages assemble compartments called phage nuclei in the bacterial cytoplasm. Thus, DNA viruses infecting host cells from different domains of life share common replication strategies.

The H/ACA snoRNP complex directs the formation of pseudouridines in ribosomal RNA (rRNA); however, the mechanisms by which the eukaryotic two-hairpin structured H/ACA snoRNP mediates pseudouridylation in rRNA are poorly understood. This study investigates the function of the conserved two-hairpin structure of eukaryotic H/ACA snoRNA while modifying large, structured rRNA. The biochemical analysis reveals that the two hairpins of the snoRNP complex can independently convert two uridines to two pseudouridines in long rRNA fragments. Moreover, the H/ACA snoRNP can likely unfold secondary structures in rRNA without the help of additional factors. Further, the GAR domains of Gar1 are required for pseudouridine formation, but the KKE/D extensions of Cbf5 play no role in pseudouridylation of rRNA. In summary, this study addresses the mechanism of the H/ACA snoRNP complex in modifying rRNA during ribosome biogenesis.

We performed a time-lapse imaging with atomic force microscopy (AFM) of the motion of eukaryotic CRFK (Crandell-Rees Feline Kidney) cells adhered onto a glass surface and anchored to other cells in culture medium at 37 °C. The main finding is a gradient in the spring constant of the actomyosin cortex along the cells axis. The rigidity increases at the rear of the cells during motion. This observation as well as a dramatic decrease of the volume suggests that cells may organize a dissymmetry in the skeleton network to expulse water and drive actively the rear edge.

The H/ACA snoRNP complex directs the formation of pseudouridines in ribosomal RNA (rRNA); however, the mechanisms by which the eukaryotic two-hairpin structured H/ACA snoRNP mediates pseudouridylation in rRNA are poorly understood. This study investigates the function of the conserved two-hairpin structure of eukaryotic H/ACA snoRNA while modifying large, structured rRNA. The biochemical analysis reveals that the two hairpins of the snoRNP complex can independently convert two uridines to two pseudouridines in long rRNA fragments. Moreover, the H/ACA snoRNP can likely unfold secondary structures in rRNA without the help of additional factors. Further, the GAR domains of Gar1 are required for pseudouridine formation, but the KKE/D extensions of Cbf5 play no role in pseudouridylation of rRNA. In summary, this study addresses the mechanism of the H/ACA snoRNP complex in modifying rRNA during ribosome biogenesis.

The H/ACA snoRNP complex directs the formation of pseudouridines in ribosomal RNA (rRNA); however, the mechanisms by which the eukaryotic two-hairpin structured H/ACA snoRNP mediates pseudouridylation in rRNA are poorly understood. This study investigates the function of the conserved two-hairpin structure of eukaryotic H/ACA snoRNA while modifying large, structured rRNA. The biochemical analysis reveals that the two hairpins of the snoRNP complex can independently convert two uridines to two pseudouridines in long rRNA fragments. Moreover, the H/ACA snoRNP can likely unfold secondary structures in rRNA without the help of additional factors. Further, the GAR domains of Gar1 are required for pseudouridine formation, but the KKE/D extensions of Cbf5 play no role in pseudouridylation of rRNA. In summary, this study addresses the mechanism of the H/ACA snoRNP complex in modifying rRNA during ribosome biogenesis.

The H/ACA snoRNP complex directs the formation of pseudouridines in ribosomal RNA (rRNA); however, the mechanisms by which the eukaryotic two-hairpin structured H/ACA snoRNP mediates pseudouridylation in rRNA are poorly understood. This study investigates the function of the conserved two-hairpin structure of eukaryotic H/ACA snoRNA while modifying large, structured rRNA. The biochemical analysis reveals that the two hairpins of the snoRNP complex can independently convert two uridines to two pseudouridines in long rRNA fragments. Moreover, the H/ACA snoRNP can likely unfold secondary structures in rRNA without the help of additional factors. Further, the GAR domains of Gar1 are required for pseudouridine formation, but the KKE/D extensions of Cbf5 play no role in pseudouridylation of rRNA. In summary, this study addresses the mechanism of the H/ACA snoRNP complex in modifying rRNA during ribosome biogenesis.

The H/ACA snoRNP complex directs the formation of pseudouridines in ribosomal RNA (rRNA); however, the mechanisms by which the eukaryotic two-hairpin structured H/ACA snoRNP mediates pseudouridylation in rRNA are poorly understood. This study investigates the function of the conserved two-hairpin structure of eukaryotic H/ACA snoRNA while modifying large, structured rRNA. The biochemical analysis reveals that the two hairpins of the snoRNP complex can independently convert two uridines to two pseudouridines in long rRNA fragments. Moreover, the H/ACA snoRNP can likely unfold secondary structures in rRNA without the help of additional factors. Further, the GAR domains of Gar1 are required for pseudouridine formation, but the KKE/D extensions of Cbf5 play no role in pseudouridylation of rRNA. In summary, this study addresses the mechanism of the H/ACA snoRNP complex in modifying rRNA during ribosome biogenesis.

type:Review Article, In general, it had been believed that the cell fate restriction of terminally differentiated somatic cells was irreversible. In 1952, somatic cell nuclear transfer (SCNT) was introduced to study early embryonic development in frogs. So far, various mammalian species have been successfully cloned using the SCNT technique, though its efficiency is very low. Embryonic stem (ES) cells were the first pluripotent cells to be isolated from an embryo and have a powerful potential to differentiate into more than 260 types of cells. The generation of induced pluripotent stem (iPS) cells was a breakthrough in stem cell research, and the use of these iPS cells has solved problems such as low efficiency and cell fate restriction. These cells have since been utilized for clinical application, disease investigation, and drug selection. As it is widely accepted that the endosymbiosis of Archaea into eukaryotic ancestors resulted in the generation of eukaryotic cells, we examined whether bacterial infection could alter host cell fate. We previously showed that when human dermal fibroblast (HDF) cells were incorporated with lactic acid bacteria (LAB), the LAB-incorporated HDF cells formed clusters and expressed a subset of common pluripotent markers. Moreover, LAB-incorporated cell clusters could differentiate into cells derived from each of the three germinal layers both in vivo and in vitro, indicating successful reprogramming of host HDF cells by LAB. In the current review, we introduce the existing examples of cellular reprogramming by bacteria and discuss their nuclear reprogramming mechanisms.

Natural compounds have been commercially used in pharmaceutical industry due to their therapeutic nature. Herein, natural compounds are screened that modulate various biological processes that are known to be conserved from yeast to human and to be associated with human diseases such as cancers and viral infections. Baker’s yeast Saccharomyces cerevisiae is a eukaryotic model in the pharmaceutical industry and its use allows genome-wide recognition of potential targets of natural compounds. Cancer is one of the leading causes of death worldwide with the number of diagnosed cases of this disease escalating. Chemotherapy remains the number one treatment option, especially in developing countries where other forms of treatment remain costly. The anthracyclines (natural compounds) are chemotherapeutic agents that have been in use for the last forty years and that are effective in treating various solid tumours as well as haematological malignancies. Various modes of action have been suggested for these agents; however, it remains unclear as to how these agents really exert their cytotoxic effects on cells. Anthracyclines can cause severe adverse effects such as cardiotoxicity as well as myelosuppression, and these are the major limitations of these drugs. To solve these problems, the controversy regarding their mechanism of action needs to be addressed. For this reason, here the focus is on the analysis of molecular mechanisms responsible for anthracycline cytotoxicity and the identity of the intracellular compartments that anthracyclines localise to in a mammalian cell line and relate the findings to those controversies found in the literature. In addition, natural compound-based potential anti-viral compounds that may inhibit cellular vacuolar protein sorting-4 (Vps4p) ATPase activity and in parallel block viral budding from the cellular membrane were also investigated. Natural compounds have been largely excluded from characterisation via high-throughput profiling st, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Pharmacy & Med Sci, Griffith Health, Full Text

In this short Perspective, we discuss how recent dynamic live-cell imaging experiments have challenged our understanding of mechanisms driving functional molecular interactions in vivo. While we have generally considered the formation of functional biomolecular complexes as resulting from the stable assembly of two or more partner molecules, here we entertain the possibility that function may actually be maintained while molecules are rapidly exchanged within a complex. We postulate that at high effective concentrations, even very weak interactions can lead to strong binding site occupancy and thereby mediate function in a highly dynamic fashion. This new perspective in our definition of what represents a functional complex in living cells and in vivo could significantly alter how we define the nature of molecular transactions critical for mediating regulation in the cellular context. These less conventional principles also allow a broadening of the mechanistic options we should explore when interpreting essential biological processes such as gene regulation.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4—a cancer insertion–deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions —is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome., We thank S. Jinks-Robertson for suggesting the traffic light reporter approach; H. Klein for guidance on fluctuation assays; R. van Boxtel for sharing sequencing data for MLH1-KO organoids; A. Bretherick, O. B. Reina and G. Kudla for advice on HygroR re-coding; staff at the IGC core services (L. Murphy, C. Nicol, C. Warnock, E. Freyer, S. Brown and J. Joseph), C. Logan, A. Fluteau, A. Robertson and the staff at Edinburgh Genomics for technical assistance; staff at Liverpool CLL Biobank (funded by Blood Cancer UK) for samples used to generate GEL WGS data; A. Ewing, C.-A. Martin, N. Hastie and W. Bickmore for discussions. Funding for this work: UK Medical Research Council Human Genetics Unit core grants (MC_UU_00007/5 to A.P.J., MC_UU_00007/11 to M.S.T.); Edinburgh Clinical Academic Track PhD programme (Wellcome Trust 204802/Z/16/Z) to T.C.W.; 2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research (grant number 21-40-11-NADE) to F.N.; a CRUK Brain Tumour Centre of Excellence Award (C157/A27589) to M.D.N.; EKFS research grant (2019_A09), Wilhelm Sander-Stiftung (2019.046.1) to K.A., CRUK programme grant (C20807/A2864) to T.S.; La Caixa Foundation (CLLEvolution-LCF/PR/HR17/52150017, Health Research 2017 Program HR17-00221) to E.C.; E.C. is an Academia Researcher of the Institució Catalana de Recerca i Estudis Avançats of the Generalitat de Catalunya. Edinburgh Genomics is partly supported by NERC (R8/H10/56), MRC (MR/K001744/1) and BBSRC (BB/J004243/1). This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provide, Peer Reviewed, "Article signat per 22 autors/es: Martin A. M. Reijns, David A. Parry, Thomas C. Williams, Ferran Nadeu, Rebecca L. Hindshaw, Diana O. Rios Szwed, Michael D. Nicholson, Paula Carroll, Shelagh Boyle, Romina Royo, Alex J. Cornish, Hang Xiang, Kate Ridout, The Genomics England Research Consortium, Colorectal Cancer Domain UK 100,000 Genomes Project, Anna Schuh, Konrad Aden, Claire Palles, Elias Campo, Tatjana Stankovic, Martin S. Taylor & Andrew P. Jackson ", Postprint (published version)

Unicellular eukaryotic predators play a crucial role in the functioning of the ocean ecosystem by recycling nutrients and energy that are channeled to upper trophic levels. Traditionally, these evolutionarily diverse organisms have been combined into a single functional group (heterotrophic flagellates), overlooking their organismal differences. Here, we investigated four evolutionarily related species belonging to one cosmopolitan group of uncultured marine picoeukaryotic predators: marine stramenopiles (MAST)-4 (species A, B, C, and E). Co-occurrence and distribution analyses in the global surface ocean indicated contrasting patterns in MAST-4A and C, suggesting adaptation to different temperatures. We then investigated whether these spatial distribution patterns were mirrored by MAST-4 genomic content using single-cell genomics. Analyses of 69 single cells recovered 66 to 83% of the MAST-4A/B/C/E genomes, which displayed substantial interspecies divergence. MAST-4 genomes were similar in terms of broad gene functional categories, but they differed in enzymes of ecological relevance, such as glycoside hydrolases (GHs), which are part of the food degradation machinery in MAST-4. Interestingly, MAST-4 species featuring a similar GH composition (A and C) coexcluded each other in the surface global ocean, while species with a different set of GHs (B and C) appeared to be able to coexist, suggesting further niche diversification associated with prey digestion. We propose that differential niche adaptation to temperature and prey type has promoted adaptive evolutionary diversification in MAST-4. We show that minute ocean predators from the same phylogenetic group may have different biogeography and genomic content, which needs to be accounted for to better comprehend marine food webs., Postprint (published version)

We advance an account that grounds cognition, specifically decision-making, in an activity all organisms as autonomous systems must perform to keep themselves viable-controlling their production mechanisms. Production mechanisms, as we characterize them, perform activities such as procuring resources from their environment, putting these resources to use to construct and repair the organism's body and moving through the environment. Given the variable nature of the environment and the continual degradation of the organism, these production mechanisms must be regulated by control mechanisms that select when a production is required and how it should be carried out. To operate on production mechanisms, control mechanisms need to procure information through measurement processes and evaluate possible actions. They are making decisions. In all organisms, these decisions are made by multiple different control mechanisms that are organized not hierarchically but heterarchically. In many cases, they employ internal models of features of the environment with which the organism must deal. Cognition, in the form of decision-making, is thus fundamental to living systems which must control their production mechanisms. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

Oxidative phosphorylation is among the most conserved mitochondrial pathways. However, one of the cornerstones of this pathway, the multi-protein complex NADH : ubiquinone oxidoreductase (complex I) has been lost multiple independent times in diverse eukaryotic lineages. The causes and consequences of these convergent losses remain poorly understood. Here, we used a comparative genomics approach to reconstruct evolutionary paths leading to complex I loss and infer possible evolutionary scenarios. By mining available mitochondrial and nuclear genomes, we identified eight independent events of mitochondrial complex I loss across eukaryotes, of which six occurred in fungal lineages. We focused on three recent loss events that affect closely related fungal species, and inferred genomic changes convergently associated with complex I loss. Based on these results, we predict novel complex I functional partners and relate the loss of complex I with the presence of increased mitochondrial antioxidants, higher fermentative capabilities, duplications of alternative dehydrogenases, loss of alternative oxidases and adaptation to antifungal compounds. To explain these findings, we hypothesize that a combination of previously acquired compensatory mechanisms and exposure to environmental triggers of oxidative stress (such as hypoxia and/or toxic chemicals) induced complex I loss in fungi., This work was supported by grants from the Spanish Ministry of Science and Innovation (grant no. PGC2018-099921-B-I00), cofounded by European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423; from the European Union's Horizon 2020 research and (grant no. ERC-2016-724173); and from the Instituto de Salud Carlos III—Instituto Nacional de Bioinformatica, (grant no. PT17/0009/0023—ISCIII-SGEFI/ERDF). M.À.S.-T. received a Predoctoral Each Fellowship from ‘Caixa’ Foundation (grant no. LCF/BQ/DR19/11740023)., Peer Reviewed, Postprint (published version)

We conducted an exhaustive search for three-dimensional structural homologs to the proteins of 20 key phylogenetically distinct nucleocytoplasmic DNA viruses (NCLDV). Structural matches covered 429 known protein domain superfamilies, with the most highly represented being ankyrin repeat, P-loop NTPase, F-box, protein kinase, and membrane occupation and recognition nexus (MORN) repeat. Domain superfamily diversity correlated with genome size, but a diversity of around 200 superfamilies appeared to correlate with an abrupt switch to paralogization. Extensive structural homology was found across the range of eukaryotic RNA polymerase II subunits and their associated basal transcription factors, with the coordinated gain and loss of clusters of subunits on a virus-by-virus basis. The total number of predicted endonucleases across the 20 NCLDV was nearly quadrupled from 36 to 132, covering much of the structural and functional diversity of endonucleases throughout the biosphere in DNA restriction, repair, and homing. Unexpected findings included capsid protein-transcription factor chimeras; endonuclease chimeras; enzymes for detoxification; antimicrobial peptides and toxin-antitoxin systems associated with symbiosis, immunity, and addiction; and novel proteins for membrane abscission and protein turnover.IMPORTANCE We extended the known annotation space for the NCLDV by 46%, revealing high-probability structural matches for fully 45% of the 9,671 query proteins and confirming up to 98% of existing annotations per virus. The most prevalent protein families included ankyrin repeat- and MORN repeat-containing proteins, many of which included an F-box, suggesting extensive host cell modulation among the NCLDV. Regression suggested a minimum requirement for around 36 protein structural superfamilies for a viable NCLDV, and beyond around 200 superfamilies, genome expansion by the acquisition of new functions was abruptly replaced by paralogization. We found homologs to herpesv

Our current knowledge about nucleocytoplasmic large DNA viruses (NCLDVs) is largely derived from viral isolates that are co-cultivated with protists and algae. Here we reconstructed 2,074 NCLDV genomes from sampling sites across the globe by building on the rapidly increasing amount of publicly available metagenome data. This led to an 11-fold increase in phylogenetic diversity and a parallel 10-fold expansion in functional diversity. Analysis of 58,023 major capsid proteins from large and giant viruses using metagenomic data revealed the global distribution patterns and cosmopolitan nature of these viruses. The discovered viral genomes encoded a wide range of proteins with putative roles in photosynthesis and diverse substrate transport processes, indicating that host reprogramming is probably a common strategy in the NCLDVs. Furthermore, inferences of horizontal gene transfer connected viral lineages to diverse eukaryotic hosts. We anticipate that the global diversity of NCLDVs that we describe here will establish giant viruses-which are associated with most major eukaryotic lineages-as important players in ecosystems across Earth's biomes.

The accuracy of DNA replication, gene transcription, and protein translation is critical to maintain the integrity of the genetic code. Mistakes that occur during transcription can cause genomic instability and human diseases. Here, we employed Circular Sequencing (Cir-seq) to investigate the mutational profiles and landscape of the yeast Saccharomyces Cerevisiae W303-1A-Rad5R535 strain that carries a variant of Rad5 gene that may impairs transcription-coupled DNA repair or promote transcription-replication conflicts. Three batches of Cir-seq are prepared with error rates of 1.99 × 10-6, 4.66 × 10-6 and 5.27× 10-6. Dominant G to U mutations are found in two batches with a high coverage number. G to U errors are clustered when the focal base of G is flanked by GU and UC. A list of hotspots is obtained for an error rate range of 10-3 to 10-6 with all types of mutations, most of which are related to the ribosomal gene, which may impact proteostasis. Background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) is our newly developed method for accurate sequencing of nascent RNA produced by RNA polymerase I. We present the initial development of EmPC-seq and identification of error hotspots and the features of the RNA polymerase I transcribed region by introducing a background baseline. Mismatch rates are greatly reduced from 1.43±0.11×10-3 (PRO-seq) to 3.79±0.81×10-5 (EmPC-seq), implying that EmPC-seq can successfully remove noise generated mainly during reverse transcription. One feature of RNA polymerase I is that errors tend to cluster at the 3’ end. EmPC-seq can be further extended to study different transcriptional error spectra of pre-mRNA (RNA polymerase II transcribed), siRNAs, and regulatory RNA between normal and disease cells. Following the development of EmPC-seq, we were inspired to develop nucleotide resolution sequencing to obtain the influenz

The accuracy of DNA replication, gene transcription, and protein translation is critical to maintain the integrity of the genetic code. Mistakes that occur during transcription can cause genomic instability and human diseases. Here, we employed Circular Sequencing (Cir-seq) to investigate the mutational profiles and landscape of the yeast Saccharomyces Cerevisiae W303-1A-Rad5R535 strain that carries a variant of Rad5 gene that may impairs transcription-coupled DNA repair or promote transcription-replication conflicts. Three batches of Cir-seq are prepared with error rates of 1.99 × 10-6, 4.66 × 10-6 and 5.27× 10-6. Dominant G to U mutations are found in two batches with a high coverage number. G to U errors are clustered when the focal base of G is flanked by GU and UC. A list of hotspots is obtained for an error rate range of 10-3 to 10-6 with all types of mutations, most of which are related to the ribosomal gene, which may impact proteostasis. Background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) is our newly developed method for accurate sequencing of nascent RNA produced by RNA polymerase I. We present the initial development of EmPC-seq and identification of error hotspots and the features of the RNA polymerase I transcribed region by introducing a background baseline. Mismatch rates are greatly reduced from 1.43±0.11×10-3 (PRO-seq) to 3.79±0.81×10-5 (EmPC-seq), implying that EmPC-seq can successfully remove noise generated mainly during reverse transcription. One feature of RNA polymerase I is that errors tend to cluster at the 3’ end. EmPC-seq can be further extended to study different transcriptional error spectra of pre-mRNA (RNA polymerase II transcribed), siRNAs, and regulatory RNA between normal and disease cells. Following the development of EmPC-seq, we were inspired to develop nucleotide resolution sequencing to obtain the influenz

Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques. This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.

The accuracy of DNA replication, gene transcription, and protein translation is critical to maintain the integrity of the genetic code. Mistakes that occur during transcription can cause genomic instability and human diseases. Here, we employed Circular Sequencing (Cir-seq) to investigate the mutational profiles and landscape of the yeast Saccharomyces Cerevisiae W303-1A-Rad5R535 strain that carries a variant of Rad5 gene that may impairs transcription-coupled DNA repair or promote transcription-replication conflicts. Three batches of Cir-seq are prepared with error rates of 1.99 × 10-6, 4.66 × 10-6 and 5.27× 10-6. Dominant G to U mutations are found in two batches with a high coverage number. G to U errors are clustered when the focal base of G is flanked by GU and UC. A list of hotspots is obtained for an error rate range of 10-3 to 10-6 with all types of mutations, most of which are related to the ribosomal gene, which may impact proteostasis. Background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) is our newly developed method for accurate sequencing of nascent RNA produced by RNA polymerase I. We present the initial development of EmPC-seq and identification of error hotspots and the features of the RNA polymerase I transcribed region by introducing a background baseline. Mismatch rates are greatly reduced from 1.43±0.11×10-3 (PRO-seq) to 3.79±0.81×10-5 (EmPC-seq), implying that EmPC-seq can successfully remove noise generated mainly during reverse transcription. One feature of RNA polymerase I is that errors tend to cluster at the 3’ end. EmPC-seq can be further extended to study different transcriptional error spectra of pre-mRNA (RNA polymerase II transcribed), siRNAs, and regulatory RNA between normal and disease cells. Following the development of EmPC-seq, we were inspired to develop nucleotide resolution sequencing to obtain the influenz

Evolution of eukaryotic species and their genomes has been traditionally understood as a vertical process in which genetic material is transmitted from parents to offspring along a lineage, and in which genetic exchange is restricted within species boundaries. However, mounting evidence from comparative genomics indicates that this paradigm is often violated. Horizontal gene transfer and mating between diverged lineages blur species boundaries and challenge the reconstruction of evolutionary histories of species and their genomes. Nonvertical evolution might be more restricted in eukaryotes than in prokaryotes, yet it is not negligible and can be common in certain groups. Recognition of such processes brings about the need to incorporate this complexity into our models, as well as to conceptually reframe eukaryotic diversity and evolution. Here, I review the recent work from genomics studies that supports the effects of nonvertical modes of evolution including introgression, hybridization, and horizontal gene transfer in different eukaryotic groups. I then discuss emerging patterns and effects, illustrated by specific examples, that support the conclusion that nonvertical processes are often at the root of important evolutionary transitions and adaptations. I will argue that a paradigm shift is needed to naturally accommodate nonvertical processes in eukaryotic evolution., The author acknowledges support from the Spanish Ministry of Science and Innovation for the Grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423, and Grants from the European Union's Horizon 2020 research and innovation programme under the Grant agreement ERC-2016-724173, and the Marie Sklodowska-Curie Grant agreement H2020-MSCA-IF-2017-793699. The author also receives support from an INB Grant (PT17/0009/0023 -ISCIII-SGEFI/ERDF)., Peer Reviewed, Postprint (published version)

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co-existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter-organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.

In response to stress, cells must quickly reprogram gene expression to adapt and survive. This is achieved in part by altering levels of mRNAs and their translation into proteins. Recently, the formation of two stress-induced messenger ribonucleoprotein (mRNP) assemblies named stress granules and processing bodies has been postulated to directly impact gene expression during stress. These assemblies sequester and concentrate specific proteins and RNAs away from the larger cytoplasm during stress, thereby providing a layer of posttranscriptional gene regulation with the potential to directly impact mRNA levels, protein translation, and cell survival. The function of these granules has generally been ascribed either by the protein components concentrated into them or, more broadly, by global changes that occur during stress. Recent proteome- and transcriptome-wide studies have provided a more complete view of stress-induced mRNP granule composition in varied cell types and stress conditions. However, direct measurements of the phenotypic and functional consequences of stress granule and processing body formation are lacking. This leaves our understanding of their roles during stress incomplete. Continued study into the function of these granules will be an important part in elucidating how cells respond to and survive stressful environmental changes. This article is categorized under: Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization.

Transfer RNAs are the largest, most complex non-coding RNA family, universal to all living organisms. tRNAscan-SE has been the de facto tool for predicting tRNA genes in whole genomes. The newly developed version 2.0 has incorporated advanced methodologies with improved probabilistic search software and a suite of new gene models, enabling better functional classification of predicted genes. This chapter describes the use of the UNIX command-driven and online web versions, illustrating different search modes and options.

Quantitative analysis of the giant ciliate Stentor roeselii shows that a single cell can make decisions, based on the ability to switch between several different behaviors in a non-random order.

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.

Single-cell genomics (SCG) methods provide a unique opportunity to analyse whole genome information at the resolution of an individual cell. While SCG has been extensively used to investigate bacterial and archaeal genomes, the technique has been rarely used to access the genetic makeup of uncultivated microbial eukaryotes. In this regard, the use of SCG can provide a wealth of information; not only do the methods allow exploration of the genome, they can also help elucidate the relationship between the cell and intracellular entities extant in nearly all eukaryotes. SCG enables the study of total eukaryotic cellular DNA, which in turn allows us to better understand the evolutionary history and diversity of life, and the physiological interactions that define complex organisms. This article is part of a discussion meeting issue 'Single cell ecology'.

Protection against microbial infection in eukaryotes is provided by diverse cellular and molecular mechanisms. Here, we present a comparative view of the antiviral activity of virus-derived small interfering RNAs in fungi, plants, invertebrates and mammals, detailing the mechanisms for their production, amplification and activity. We also highlight the recent discovery of viral PIWI-interacting RNAs in animals and a new role for mobile host and pathogen small RNAs in plant defence against eukaryotic pathogens. In turn, viruses that infect plants, insects and mammals, as well as eukaryotic pathogens of plants, have evolved specific virulence proteins that suppress RNA interference (RNAi). Together, these advances suggest that an antimicrobial function of the RNAi pathway is conserved across eukaryotic kingdoms.

Natural products that target the eukaryotic ribosome are promising therapeutics to treat a variety of cancers. It is therefore essential to determine their molecular mechanism of action to fully understand their mode of interaction with the target and to inform the development of new synthetic compounds with improved potency and reduced cytotoxicity. Toward this goal, we have previously established a short synthesis pathway that grants access to multiple congeners of the lissoclimide family. Here we present the X-ray co-crystal structure at 3.1 Å resolution of C45, a potent congener with two A-ring chlorine-bearing stereogenic centers with 'unnatural' configurations, with the yeast 80S ribosome, intermolecular interaction energies of the C45/ribosome complex, and single-molecule FRET data quantifying the impact of C45 on both human and yeast ribosomes. Together, these data provide new insights into the role of unusual non-covalent halogen bonding interactions involved in the binding of this synthetic compound to the 80S ribosome.

Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.

Understanding how biodiversity changes in time and space is vital to assess the effects of environmental change on benthic ecosystems. Due to the limitations of morphological methods, there has been a rapid expansion in the application of high-throughput sequencing methods to study benthic eukaryotic communities. However, the effect of sample size and small-scale spatial variation on the assessment of benthic eukaryotic diversity is still not well understood. Here, we investigate the effect of different sample volumes in the genetic assessment of benthic metazoan and non-metazoan eukaryotic community composition. Accordingly, DNA was extracted from five different cumulative sediment volumes comprising 100% of the top 2 cm of five benthic sampling cores, and used as template for Ilumina MiSeq sequencing of 18 S rRNA amplicons. Sample volumes strongly impacted diversity metrics for both metazoans and non-metazoan eukaryotes. Beta-diversity of treatments using smaller sample volumes was significantly different from the beta-diversity of the 100% sampled area. Overall our findings indicate that sample volumes of 0.2 g (1% of the sampled area) are insufficient to account for spatial heterogeneity at small spatial scales, and that relatively large percentages of sediment core samples are needed for obtaining robust diversity measurement of both metazoan and non-metazoan eukaryotes.

All messenger RNAs in eukaryotes are modified co-transcriptionally and post-transcriptionally. They are all capped at the 5'-end and polyadenylated at the 3'-end. However, many mRNAs are also found to be chemically modified internally for regulation of mRNA processing, translation, stability, and to recode the message. This review will briefly summarize the structural basis for formation of the two most common modifications found at internal sites in mRNAs; methylation and deamination. The structures of the enzymes that catalyze these modifications show structural similarity to other family members within each modifying enzyme class. RNA methyltransferases, including METTL3/METTL14 responsible for N6-methyladensosine (m6A) formation, share a common structural core and utilize S-adenosyl methionine as a methyl donor. RNA deaminases, including adenosine deaminases acting on RNA (ADARs), also share a common structural core and similar signature sequence motif with conserved residues used for binding zinc and catalyzing the deamination reaction. In spite of recent reports of high resolution structures for members of these two RNA-modifying enzyme families, a great deal remains to be uncovered for a complete understanding of the structural basis for mRNA modification. Of particular interest is the definition of factors that control modification site specificity.

Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes.

Cells in tissues or biofilms communicate with one another through chemical and mechanical signals to coordinate collective behaviors. Non-living cell mimics provide simplified models of natural systems; however, it has remained challenging to implement communication capabilities comparable to living cells. Here we present a porous artificial cell-mimic containing a nucleus-like DNA-hydrogel compartment that is able to express and display proteins, and communicate with neighboring cell-mimics through diffusive protein signals. We show that communication between cell-mimics allows distribution of tasks, quorum sensing, and cellular differentiation according to local environment. Cell-mimics can be manufactured in large quantities, easily stored, chemically modified, and spatially organized into diffusively connected tissue-like arrangements, offering a means for studying communication in large ensembles of artificial cells.