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John M Zajecka,1,2 Arielle D Stanford,3 Asli Memisoglu,4 William F Martin,5 Sanjeev Pathak3 1Department of Psychiatry, Rush University Medical Center, Chicago, IL, USA; 2Psychiatric Medicine Associates, LLC, Skokie, IL, USA; 3Department of Clinical Research, Alkermes, Inc., Waltham, MA, USA; 4Department of Biostatistics, Alkermes, Inc., Waltham, MA, USA; 5Clinical Operations, Alkermes, Inc., Waltham, MA, USA Background: The endogenous opioid system is a fundamental regulator of mood in humans. Previously reported clinical trials have demonstrated the efficacy of the investigational agent buprenorphine/samidorphan (BUP/SAM) combination, an opioid-system modulator, for the adjunctive treatment of major depressive disorder. We present here a third phase III study of different design. Methods: Adult patients with major depressive disorder and inadequate response to antidepressant therapy were enrolled in this double-blind, placebo-controlled, placebo run-in study to evaluate the efficacy, safety, and tolerability of adjunctive BUP/SAM 2 mg/2 mg. Patients with baseline Hamilton Depression Rating Scale score ≥20 received double-blind placebo in addition to background antidepressant therapy for 4 weeks. Nonresponders were randomized to receive adjunctive BUP/SAM 2 mg/2 mg or placebo for 6 weeks. The primary end point was change in Montgomery–Åsberg Depression Rating Scale (MADRS)-10 total score from randomization at baseline to the end of the 6-week treatment period.Results: Least-squares mean change in MADRS-10 score at end of treatment was -4.8 (SE 0.67) in the BUP/SAM 2 mg/2 mg group and -4.6 (SE 0.66) in the placebo group (mean difference -0.3 [SE 0.95], P=0.782). There were no differences in MADRS-based response or remission rates. Overall, 42.9% of the BUP/SAM 2 mg/2 mg group and 34.5% of the placebo group experienced at least one treatment-emergent adverse event during the 6-week treatment period, most of which were mild or moderate in severity. There were no clinically important changes in laboratory parameters, weight, or vital signs and no evidence of abuse potential during treatment or opiate-withdrawal symptoms post treatment.Conclusion: Efficacy results in FORWARD-3 measured by change in MADRS-10 score did not meet the primary end point, but postbaseline improvement in MADRS-10 in the BUP/SAM 2 mg/2 mg group was consistent with that seen in previously reported trials. BUP/SAM 2 mg/2 mg was well tolerated. Keywords: buprenorphine, samidorphan, randomized clinical trial, adjunctive therapy, study design, placebo response, opioid system modulator

Current views of O 2 accumulation in Earth history depict three phases: The onset of O 2 production by ∼2.4 billion years ago; 2 billion years of stasis at ∼1 % of modern atmospheric levels; and a rising phase, starting about 500 million years ago, in which oxygen eventually reached modern values. Purely geochemical mechanisms have been proposed to account for this tripartite time course of Earth oxygenation. In particular the second phase, the long period of stasis between the advent of O 2 and the late rise to modern levels, has posed a puzzle. Proposed solutions involve Earth processes (geochemical, ecosystem, day length). Here we suggest that Earth oxygenation was not determined by geochemical processes. Rather it resulted from emergent biological innovations associated with photosynthesis and the activity of only three enzymes: 1) The oxygen evolving complex of cyanobacteria that makes O 2 ; 2) Nitrogenase, with its inhibition by O 2 causing two billion years of oxygen level stasis; 3) Cellulose synthase of land plants, which caused mass deposition and burial of carbon, thus removing an oxygen sink and therefore increasing atmospheric O 2 . These three enzymes are endogenously produced by, and contained within, cells that have the capacity for exponential growth. The catalytic properties of these three enzymes paved the path of Earth's atmospheric oxygenation, requiring no help from Earth other than the provision of water, CO 2 , salts, colonizable habitats, and sunlight., 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 © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Life is an exergonic chemical reaction. Many individual reactions in metabolism entail slightly endergonic steps that are coupled to free energy release, typically as ATP hydrolysis, in order to go forward. ATP is almost always supplied by the rotor-stator ATP synthase, which harnesses chemiosmotic ion gradients. Because the ATP synthase is a protein, it arose after the ribosome did. What was the energy currency of metabolism before the origin of the ATP synthase and how (and why) did ATP come to be the universal energy currency? About 27 % of a cell's energy budget is consumed as GTP during translation. The universality of GTP-dependence in ribosome function indicates that GTP was the ancestral energy currency of protein synthesis. The use of GTP in translation and ATP in small molecule synthesis are conserved across all lineages, representing energetic compartments that arose in the last universal common ancestor, LUCA. And what came before GTP? Recent findings indicate that the energy supporting the origin of LUCA's metabolism stemmed from H 2 -dependent CO 2 reduction along routes that strongly resemble the reactions and transition metal catalysts of the acetyl-CoA pathway., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

ConspectusLife is an exergonic chemical reaction. The same was true when the very first cells emerged at life's origin. In order to live, all cells need a source of carbon, energy, and electrons to drive their overall reaction network (metabolism). In most cells, these are separate pathways. There is only one biochemical pathway that serves all three needs simultaneously: the acetyl-CoA pathway of CO 2 fixation. In the acetyl-CoA pathway, electrons from H 2 reduce CO 2 to pyruvate for carbon supply, while methane or acetate synthesis are coupled to energy conservation as ATP. This simplicity and thermodynamic favorability prompted Georg Fuchs and Erhard Stupperich to propose in 1985 that the acetyl-CoA pathway might mark the origin of metabolism, at the same time that Steve Ragsdale and Harland Wood were uncovering catalytic roles for Fe, Co, and Ni in the enzymes of the pathway. Subsequent work has provided strong support for those proposals.In the presence of Fe, Co, and Ni in their native metallic state as catalysts, aqueous H 2 and CO 2 react specifically to formate, acetate, methane, and pyruvate overnight at 100 °C. These metals (and their alloys) thus replace the function of over 120 enzymes required for the conversion of H 2 and CO 2 to pyruvate via the pathway and its cofactors, an unprecedented set of findings in the study of biochemical evolution. The reactions require alkaline conditions, which promote hydrogen oxidation by proton removal and are naturally generated in serpentinizing (H 2 -producing) hydrothermal vents. Serpentinizing hydrothermal vents furthermore produce natural deposits of native Fe, Co, Ni, and their alloys. These are precisely the metals that reduce CO 2 with H 2 in the laboratory; they are also the metals found at the active sites of enzymes in the acetyl-CoA pathway. Iron, cobalt and nickel are relicts of the environments in which metabolism arose, environments that still harbor ancient methane- and acetate-producing autotrophs today. This convergence indicates bedrock-level antiquity for the acetyl-CoA pathway. In acetogens and methanogens growing on H 2 as reductant, the acetyl-CoA pathway requires flavin-based electron bifurcation as a source of reduced ferredoxin (a 4Fe4S cluster-containing protein) in order to function. Recent findings show that H 2 can reduce the 4Fe4S clusters of ferredoxin in the presence of native iron, uncovering an evolutionary precursor of flavin-based electron bifurcation and suggesting an origin of FeS-dependent electron transfer in proteins. Traditionally discussed as catalysts in early evolution, the most common function of FeS clusters in metabolism is one-electron transfer, also in radical SAM enzymes, a large and ancient enzyme family. The cofactors and active sites in enzymes of the acetyl-CoA pathway uncover chemical antiquity in metabolism involving metals, methyl groups, methyl transfer reactions, cobamides, pterins, GTP, S -adenosylmethionine, radical SAM enzymes, and carbon-metal bonds. The reaction sequence from H 2 and CO 2 to pyruvate on naturally deposited native metals is maximally simple. It requires neither nitrogen, sulfur, phosphorus, RNA, ion gradients, nor light. Solid-state metal catalysts tether the origin of metabolism to a H 2 -producing, serpentinizing hydrothermal vent.

The COVID-19 pandemic has pushed the population to adopt increasingly sedentary lifestyles. Faced with this problem, remote training appears as a practical and inexpensive strategy to promote physically active lifestyles. The aim of this research was to compare the effects of remote versus in-person training on metabolic profiles and body composition of physically inactive adults. This research was conducted through a randomized, single-blind clinical trial with balanced block randomization. The sample consisted of 30 physically inactive subjects of both sexes between 18 and 30 years of age. The sample was selected using a voluntary public call. The 30 subjects were randomized into three groups of 10 people each. One group trained for 36 sessions remotely, and the other did so in person. The control group did not have a training plan. The variables evaluated pre- and post-intervention were body composition by bioimpedance, grip strength through dynamometry, primary outcome, and metabolic profile assessed from a capillary sample using the CARDIOCHEK equipment. In the remote training group, significant gains were evident in the variables of weight ( p = 0.042, d = 1.119), muscle percentage ( p = 0.032, d = 0.499), and fat percentage ( p = 0.001, d = 1.132), visceral fat ( p = 0.032, d = 0.424), total cholesterol ( p = 0.001, d = 1.213), HDL ( p = 0.001, d = 0.534), LDL ( p = 0.001, d = 0.973), triglycerides ( p = 0.001, d = 0.583), and grip strength ( p = 0.001, d = 1.201). When comparing the effects between the remote and in-person training groups, it is evident that the improvements were similar in all variables, except for glucose, in which the in-person group had a greater value reduction.

Molecular oxygen is a stable diradical. All O 2 -dependent enzymes employ a radical mechanism. Generated by cyanobacteria, O 2 started accumulating on Earth 2.4 billion years ago. Its evolutionary impact is traditionally sought in respiration and energy yield. We mapped 365 O 2 -dependent enzymatic reactions of prokaryotes to phylogenies for the corresponding 792 protein families. The main physiological adaptations imparted by O 2 -dependent enzymes were not energy conservation, but novel organic substrate oxidations and O 2 -dependent, hence O 2 -tolerant, alternative pathways for O 2 -inhibited reactions. Oxygen-dependent enzymes evolved in ancestrally anaerobic pathways for essential cofactor biosynthesis including NAD + , pyridoxal, thiamine, ubiquinone, cobalamin, heme, and chlorophyll. These innovations allowed prokaryotes to synthesize essential cofactors in O 2 -containing environments, a prerequisite for the later emergence of aerobic respiratory chains., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Hydrogen (H 2 ) has powered microbial metabolism for roughly 4 billion years. The recent discovery that it also fuels geochemical analogs of the most ancient biological carbon fixation pathway sheds light on the origin of metabolism. However, it remains unclear whether H 2 can sustain more complex nonenzymatic reaction networks. Here, we show that H 2 drives the nonenzymatic reductive amination of six biological ketoacids and glyoxylate to give the corresponding amino acids in good yields using ammonium concentrations ranging from 6 to 150 mM. Catalytic amounts of nickel or ground meteorites enable these reactions at 22°C and pH 8. The same conditions promote an H 2 -dependent ketoacid-forming reductive aldol chemistry that co-occurs with reductive amination, producing a continuous reaction network resembling amino acid synthesis in the metabolic core of ancient microbes. The results support the hypothesis that the earliest biochemical networks could have emerged without enzymes or RNA., Competing Interests: Declaration of Interests The authors declare no competing interests.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO 2 and were chemolithoautotrophs that obtained their energy and electrons from H 2 . The acetyl-CoA pathway of CO 2 fixation is central to that view because of its antiquity: Among known CO 2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H 2 . How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe 0 ), but not Ni 0 , Co 0 , Mo 0 , NiFe, Ni 2 Fe, Ni 3 Fe, or Fe 3 O 4 , promotes the H 2 -dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H 2 -dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today., Competing Interests: Competing interests statement:The authors declare no competing interest.

Humans, like all mammals, depend on the gut microbiome for digestion of cellulose, the main component of plant fiber. However, evidence for cellulose fermentation in the human gut is scarce. We have identified ruminococcal species in the gut microbiota of human populations that assemble functional multienzymatic cellulosome structures capable of degrading plant cell wall polysaccharides. One of these species, which is strongly associated with humans, likely originated in the ruminant gut and was subsequently transferred to the human gut, potentially during domestication where it underwent diversification and diet-related adaptation through the acquisition of genes from other gut microbes. Collectively, these species are abundant and widespread among ancient humans, hunter-gatherers, and rural populations but are rare in populations from industrialized societies thus indicating potential disappearance in response to the westernized lifestyle.

To test the ability of geochemical surfaces in serpentinizing hydrothermal systems to catalyze reactions from which metabolism arose, we investigated H 2 -dependent CO 2 reduction toward metabolic intermediates over silica-supported Co-Fe catalysts. Supported catalysts converted CO 2 to various products at 180 °C and 2.0 MPa. The liquid product phase included formate, acetate, and ethanol, while the gaseous product phase consisted of CH 4 , CO, methanol, and C 2 -C 7 linear hydrocarbons. The 1/1 ratio CoFe alloy with the same composition as the natural mineral wairauite yielded the highest concentrations of formate (6.0 mM) and acetate (0.8 mM), which are key intermediates in the acetyl-coenzyme A (acetyl-CoA) pathway of CO 2 fixation. While Co-rich catalysts were proficient at hydrogenation, yielding mostly CH 4 , Fe-rich catalysts favored the formation of CO and methanol. Mechanistic studies indicated intermediate hydrogenation and C-C coupling activities of alloyed CoFe, in contrast to physical mixtures of both metals. Co in the active site of Co-Fe catalysts performed a similar reaction as tetrapyrrole-coordinated Co in the corrinoid iron-sulfur (CoFeS) methyl transferase in the acetyl-CoA pathway. In a temperature range characteristic for deeper regions of serpentinizing systems, oxygenate product formation was favored at lower, more biocompatible temperatures., Competing Interests: Conflict of Interests The authors declare no conflict of interest.

Prokaryotic genomes constantly undergo gene flux via lateral gene transfer, generating a pangenome structure consisting of a conserved core genome surrounded by a more variable accessory genome shell. Over time, flux generates change in genome content. Here, we measure and compare the rate of genome flux for 5655 prokaryotic genomes as a function of amino acid sequence divergence in 36 universally distributed proteins of the informational core (IC). We find a clock of gene content change. The long-term average rate of gene content flux is remarkably constant across all higher prokaryotic taxa sampled, whereby the size of the accessory genome-the proportion of the genome harboring gene content difference for genome pairs-varies across taxa. The proportion of species-level accessory genes per genome, varies from 0% (Chlamydia) to 30%-33% (Alphaproteobacteria, Gammaproteobacteria, and Clostridia). A clock-like rate of gene content change across all prokaryotic taxa sampled suggest that pangenome structure is a general feature of prokaryotic genomes and that it has been in existence since the divergence of bacteria and archaea., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The Moon-forming impact vaporized part of Earth's mantle, and turned the rest into a magma ocean, from which carbon dioxide degassed into the atmosphere, where it stayed until water rained out to form the oceans. The rain dissolved CO 2 and made it available to react with transition metal catalysts in the Earth's crust so as to ultimately generate the organic compounds that form the backbone of microbial metabolism. The Moon-forming impact was key in building a planet with the capacity to generate life in that it converted carbon on Earth into a homogeneous and accessible substrate for organic synthesis. Today all ecosystems, without exception, depend upon primary producers, organisms that fix CO 2 . According to theories of autotrophic origin, it has always been that way, because autotrophic theories posit that the first forms of life generated all the molecules needed to build a cell from CO 2 , forging a direct line of continuity between Earth's initial CO 2 -rich atmosphere and the first microorganisms. By modern accounts these were chemolithoautotrophic archaea and bacteria that initially colonized the crust and still inhabit that environment today., (© 2023 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Serpentinization in hydrothermal vents is central to some autotrophic theories for the origin of life because it generates compartments, reductants, catalysts and gradients. During the process of serpentinization, water circulates through hydrothermal systems in the crust where it oxidizes Fe (II) in ultramafic minerals to generate Fe (III) minerals and H 2 . Molecular hydrogen can, in turn, serve as a freely diffusible source of electrons for the reduction of CO 2 to organic compounds, provided that suitable catalysts are present. Using catalysts that are naturally synthesized in hydrothermal vents during serpentinization H 2 reduces CO 2 to formate, acetate, pyruvate, and methane. These compounds represent the backbone of microbial carbon and energy metabolism in acetogens and methanogens, strictly anaerobic chemolithoautotrophs that use the acetyl-CoA pathway of CO 2 fixation and that inhabit serpentinizing environments today. Serpentinization generates reduced carbon, nitrogen and - as newer findings suggest - reduced phosphorous compounds that were likely conducive to the origins process. In addition, it gives rise to inorganic microcompartments and proton gradients of the right polarity and of sufficient magnitude to support chemiosmotic ATP synthesis by the rotor-stator ATP synthase. This would help to explain why the principle of chemiosmotic energy harnessing is more conserved (older) than the machinery to generate ion gradients via pumping coupled to exergonic chemical reactions, which in the case of acetogens and methanogens involve H 2 -dependent CO 2 reduction. Serpentinizing systems exist in terrestrial and deep ocean environments. On the early Earth they were probably more abundant than today. There is evidence that serpentinization once occurred on Mars and is likely still occurring on Saturn's icy moon Enceladus, providing a perspective on serpentinization as a source of reductants, catalysts and chemical disequilibrium for life on other worlds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schwander, Brabender, Mrnjavac, Wimmer, Preiner and Martin.)

Formamide can serve as a key building block for the synthesis of organic molecules relevant to premetabolic processes. Natural pathways for its synthesis from CO 2 under early earth conditions are lacking. Here, we report the thermocatalytic conversion of CO 2 and H 2 O to formate and formamide over Ni-Fe nitride heterostructures in the absence of synthetic H 2 and N 2 under mild hydrothermal conditions. While water molecules act as both a solvent and hydrogen source, metal nitrides serve as nitrogen sources to produce formamide in the temperature range of 25-100 °C under 5-50 bar. Longer reaction times promote the C-C bond coupling and formation of acetate and acetamide as additional products. Besides liquid products, methane and ethane are also produced as gas-phase products. Postreaction characterization of Ni-Fe nitride particles reveals structural alteration and provides insights into the potential reaction mechanism. The findings indicate that gaseous CO 2 can serve as a carbon source for the formation of C-N bonds in formamide and acetamide over the Ni-Fe nitride heterostructure under simulated hydrothermal vent conditions.

Genes for cardiolipin and ceramide synthesis occur in some alphaproteobacterial genomes. They shed light on mitochondrial origin and signaling in the first eukaryotic cells.

Abiotic synthesis of formate and short hydrocarbons takes place in serpentinizing vents where some members of vent microbial communities live on abiotic formate as their main carbon source. To better understand the catalytic properties of Ni-Fe minerals that naturally exist in hydrothermal vents, we have investigated the ability of synthetic Ni-Fe based nanoparticular solids to catalyze the H 2 -dependent reduction of CO 2 , the first step required for the beginning of pre-biotic chemistry. Mono and bimetallic Ni-Fe nanoparticles with varied Ni-to-Fe ratios transform CO 2 and H 2 into intermediates and products of the acetyl-coenzyme A pathway-formate, acetate, and pyruvate-in mM range under mild hydrothermal conditions. Furthermore, Ni-Fe catalysts converted CO 2 to similar products without molecular H 2 by using water as a hydrogen source. Both CO 2 chemisorption analysis and post-reaction characterization of materials indicate that Ni and Fe metals play complementary roles for CO 2 fixation., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

All eukaryotes have linear chromosomes that are distributed to daughter nuclei during mitotic division, but the ancestral state of nuclear division in the last eukaryotic common ancestor (LECA) is so far unresolved. To address this issue, we have employed ancestral state reconstructions for mitotic states that can be found across the eukaryotic tree concerning the intactness of the nuclear envelope during mitosis (open or closed), the position of spindles (intranuclear or extranuclear), and the symmetry of spindles being either axial (orthomitosis) or bilateral (pleuromitosis). The data indicate that the LECA possessed closed orthomitosis with intranuclear spindles. Our reconstruction is compatible with recent findings indicating a syncytial state of the LECA, because it decouples three main processes: chromosome division, chromosome partitioning, and cell division (cytokinesis). The possession of closed mitosis using intranuclear spindles adds to the number of cellular traits that can now be attributed to LECA, providing insights into the lifestyle of this otherwise elusive biological entity at the origin of eukaryotic cells. Closed mitosis in a syncytial eukaryotic common ancestor would buffer mutations arising at the origin of mitotic division by allowing nuclei with viable chromosome sets to complement defective nuclei via mRNA in the cytosol., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The chemical reactions that formed the building blocks of life at origins required catalysts, whereby the nature of those catalysts influenced the type of products that accumulated. Recent investigations have shown that at 100 °C awaruite, a Ni 3 Fe alloy that naturally occurs in serpentinizing systems, is an efficient catalyst for CO 2 conversion to formate, acetate, and pyruvate. These products are identical with the intermediates and products of the acetyl-CoA pathway, the most ancient CO 2 fixation pathway and the backbone of carbon metabolism in H 2 -dependent autotrophic microbes. Here, we show that Ni 3 Fe nanoparticles prepared via the hard-templating method catalyze the conversion of H 2 and CO 2 to formate, acetate and pyruvate at 25 °C under 25 bar. Furthermore, the 13 C-labeled pyruvate can be further converted to acetate, parapyruvate, and citramalate over Ni, Fe, and Ni 3 Fe nanoparticles at room temperature within one hour. These findings strongly suggest that awaruite can catalyze both the formation of citramalate, the C5 product of pyruvate condensation with acetyl-CoA in microbial carbon metabolism, from pyruvate and the formation of pyruvate from CO 2 at very moderate reaction conditions without organic catalysts. These results align well with theories for an autotrophic origin of microbial metabolism under hydrothermal vent conditions., (© 2023. The Author(s).)

Serpentinizing hydrothermal systems generate H 2 as a reductant and harbor catalysts conducive to geochemical CO 2 conversion into reduced carbon compounds that form the core of microbial autotrophic metabolism. This study characterizes mineral catalysts at hydrothermal vents by investigating the interactions between catalytically active cobalt sites and silica-based support materials on H 2 -dependent CO 2 reduction. Heteroatom incorporated (Mg, Al, Ca, Ti, and Zr), ordered mesoporous silicas are applied as model support systems for the cobalt-based catalysts. It is demonstrated that all catalysts surveyed convert CO 2 to methane, methanol, carbon monoxide, and low-molecular-weight hydrocarbons at 180 °C and 20 bar, but with different activity and selectivity depending on the support modification. The additional analysis of the condensed product phase reveals the formation of oxygenates such as formate and acetate, which are key intermediates in the ancient acetyl-coenzyme A pathway of carbon metabolism. The Ti-incorporated catalyst yielded the highest concentrations of formate (3.6 mM) and acetate (1.2 mM) in the liquid phase. Chemisorption experiments including H 2 temperature-programmed reduction (TPR) and CO 2 temperature-programmed desorption (TPD) in agreement with density functional theory (DFT) calculations of the adsorption energy of CO 2 suggest metallic cobalt as the preferential adsorption site for CO 2 compared to hardly reducible cobalt-metal oxide interface species. The ratios of the respective cobalt species vary depending on the interaction strength with the support materials. The findings reveal robust and biologically relevant catalytic activities of silica-based transition metal minerals in H 2 -rich CO 2 fixation, in line with the idea that autotrophic metabolism emerged at hydrothermal vents.

Oxygen sensing mechanisms are essential for metazoans, their origin and evolution in the context of oxygen in Earth history are of interest. To trace the evolution of a main oxygen sensing mechanism among metazoans, the hypoxia induced factor, HIF, we investigated the phylogenetic distribution and phylogeny of 11 of its components across 566 eukaryote genomes. The HIF based oxygen sensing machinery in eukaryotes can be traced as far back as 800 million years (Ma) ago, likely to the last metazoan common ancestor (LMCA), and arose at a time when the atmospheric oxygen content corresponded roughly to the Pasteur point, or roughly 1% of present atmospheric level (PAL). By the time of the Cambrian explosion (541-485 Ma) as oxygen levels started to approach those of the modern atmosphere, the HIF system with its key components HIF1α, HIF1β, PHD1, PHD4, FIH and VHL was well established across metazoan lineages. HIF1α is more widely distributed and therefore may have evolved earlier than HIF2α and HIF3α, and HIF1β and is more widely distributed than HIF2β in invertebrates. PHD1, PHD4, FIH, and VHL appear in all 13 metazoan phyla. The O 2 consuming enzymes of the pathway, PHDs and FIH, have a lower substrate affinity, K m , for O 2 than terminal oxidases in the mitochondrial respiratory chain, in line with their function as an environmental signal to switch to anaerobic energy metabolic pathways. The ancient HIF system has been conserved and widespread during the period when metazoans evolved and diversified together with O 2 during Earth history., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Song, Modjewski, Kapust, Mizrahi and Martin.)

Archaea have diverse cell wall types, yet none are identical to bacterial peptidoglycan (murein). Methanogens Methanobacteria and Methanopyrus possess cell walls of pseudomurein, a structural analogue of murein. Pseudomurein differs from murein in containing the unique archaeal sugar N -acetyltalosaminuronic acid instead of N -acetylmuramic acid, β-1,3 glycosidic bonds in place of β-1,4 bonds and only l-amino acids in the peptide cross-links. We have determined crystal structures of methanogen pseudomurein peptide ligases (termed pMurE) from Methanothermus fervidus (Mfer762) and Methanothermobacter thermautotrophicus (Mth734) that are structurally most closely related to bacterial MurE peptide ligases. The homology of the archaeal pMurE and bacterial MurE enzymes is clear both in the overall structure and at the level of each of the three domains. In addition, we identified two UDP-binding sites in Mfer762 pMurE, one at the exterior surface of the interface of the N-terminal and middle domains, and a second site at an inner surface continuous with the highly conserved interface of the three domains. Residues involved in ATP binding in MurE are conserved in pMurE, suggesting that a similar ATP-binding pocket is present at the interface of the middle and the C-terminal domains of pMurE. The presence of pMurE ligases in members of the Methanobacteriales and Methanopyrales, that are structurally related to bacterial MurE ligases, supports the idea that the biosynthetic origins of archaeal pseudomurein and bacterial peptidoglycan cell walls are evolutionarily related.

The rooting of phylogenetic trees permits important inferences about ancestral states and the polarity of evolutionary events. Recently, methods that reconcile discordance between gene-trees and species-trees-tree reconciliation methods-are becoming increasingly popular for rooting species trees. Rooting via reconciliation requires values for a particular parameter, the gene transfer to gene duplication ratio (T:D), which in current practice is estimated on the fly from discordances observed in the trees. To date, the accuracy of T:D estimates obtained by reconciliation analyses has not been compared to T:D estimates obtained by independent means, hence the effect of T:D upon inferences of species tree roots is altogether unexplored. Here we investigated the issue in detail by performing tree reconciliations of more than 10,000 gene trees under a variety of T:D ratios for two phylogenetic cases: a bacterial (prokaryotic) tree with 265 species and a fungal-metazoan (eukaryotic) tree with 31 species. We show that the T:D ratios automatically estimated by a current tree reconciliation method, ALE, generate virtually identical T:D ratios across bacterial genes and fungal-metazoan genes. The T:D ratios estimated by ALE differ 10- to 100-fold from robust, ALE-independent estimates from real data. More important is our finding that the root inferences using ALE in both datasets are strongly dependent upon T:D. Using more realistic T:D ratios, the number of roots inferred by ALE consistently increases and, in some cases, clearly incorrect roots are inferred. Furthermore, our analyses reveal that gene duplications have a far greater impact on ALE's preferences for phylogenetic root placement than gene transfers or gene losses do. Overall, we show that obtaining reliable species tree roots with ALE is only possible when gene duplications are abundant in the data and the number of falsely inferred gene duplications is low. Finding a sufficient sample of true gene duplications for rooting species trees critically depends on the T:D ratios used in the analyses. T:D ratios, while being important parameters of genome evolution in their own right, affect the root inferences with tree reconciliations to an unanticipated degree.

Hydrogen gas, H 2 , is generated in serpentinizing hydrothermal systems, where it has supplied electrons and energy for microbial communities since there was liquid water on Earth. In modern metabolism, H 2 is converted by hydrogenases into organically bound hydrides (H - ), for example, the cofactor NADH. It transfers hydrides among molecules, serving as an activated and biologically harnessed form of H 2 . In serpentinizing systems, minerals can also bind hydrides and could, in principle, have acted as inorganic hydride donors-possibly as a geochemical protoenzyme, a 'geozyme'- at the origin of metabolism. To test this idea, we investigated the ability of H 2 to reduce NAD + in the presence of iron (Fe), cobalt (Co) and nickel (Ni), metals that occur in serpentinizing systems. In the presence of H 2 , all three metals specifically reduce NAD + to the biologically relevant form, 1,4-NADH, with up to 100% conversion rates within a few hours under alkaline aqueous conditions at 40 °C. Using Henry's law, the partial pressure of H 2 in our reactions corresponds to 3.6 mm, a concentration observed in many modern serpentinizing systems. While the reduction of NAD + by Ni is strictly H 2 -dependent, experiments in heavy water ( 2 H 2 O) indicate that native Fe can reduce NAD + both with and without H 2 . The results establish a mechanistic connection between abiotic and biotic hydride donors, indicating that geochemically catalysed, H 2 -dependent NAD + reduction could have preceded the hydrogenase-dependent reaction in evolution., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Two main theories have been put forward to explain the origin of mitochondria in eukaryotes: phagotrophic engulfment (undigested food) and microbial symbiosis (physiological interactions). The two theories generate mutually exclusive predictions about the order in which mitochondria and phagocytosis arose. To discriminate the alternatives, we have employed ancestral state reconstructions (ASR) for phagocytosis as a trait, phagotrophy as a feeding habit, the presence of mitochondria, the presence of plastids, and the multinucleated organization across major eukaryotic lineages. To mitigate the bias introduced by assuming a particular eukaryotic phylogeny, we reconstructed the appearance of these traits across 1789 different rooted gene trees, each having species from opisthokonts, mycetozoa, hacrobia, excavate, archeplastida, and Stramenopiles, Alveolates and Rhizaria. The trees reflect conflicting relationships and different positions of the root. We employed a novel phylogenomic test that summarizes ASR across trees which reconstructs a last eukaryotic common ancestor that possessed mitochondria, was multinucleated, lacked plastids, and was non-phagotrophic as well as non-phagocytic. This indicates that both phagocytosis and phagotrophy arose subsequent to the origin of mitochondria, consistent with findings from comparative physiology. Furthermore, our ASRs uncovered multiple origins of phagocytosis and of phagotrophy across eukaryotes, indicating that, like wings in animals, these traits are useful but neither ancestral nor homologous across groups. The data indicate that mitochondria preceded the origin of phagocytosis, such that phagocytosis cannot have been the mechanism by which mitochondria were acquired., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

A combination of olanzapine and samidorphan (OLZ/SAM) received US Food and Drug Administration approval in May 2021 for the treatment of adults with schizophrenia or bipolar I disorder. OLZ/SAM provides the efficacy of olanzapine, while mitigating olanzapine-associated weight gain. This exploratory study characterized the metabolic profile of OLZ/SAM in healthy volunteers to gain mechanistic insights. Volunteers received once-daily oral 10 mg/10 mg OLZ/SAM, 10 mg olanzapine, or placebo for 21 days. Assessments included insulin sensitivity during an oral glucose tolerance test (OGTT), hyperinsulinemic-euglycemic clamp, other measures of glucose/lipid metabolism, and adverse event (AE) monitoring. Treatment effects were estimated with analysis of covariance. In total, 60 subjects were randomized (double-blind; placebo, n = 12; olanzapine, n = 24; OLZ/SAM, n = 24). Olanzapine resulted in hyperinsulinemia and reduced insulin sensitivity during an OGTT at day 19, changes not observed with OLZ/SAM or placebo. Insulin sensitivity, measured by hyperinsulinemic-euglycemic clamp, was decreased in all treatment groups relative to baseline, but this effect was greatest with olanzapine and OLZ/SAM. Although postprandial (OGTT) glucose and fasting cholesterol concentrations were similarly increased with olanzapine or OLZ/SAM, other early metabolic effects were distinct, including post-OGTT C-peptide concentrations and aspects of energy metabolism. Forty-nine subjects (81.7%) experienced at least 1 AE, most mild or moderate in severity. OLZ/SAM appeared to mitigate some of olanzapine's unfavorable postprandial metabolic effects (e.g., hyperinsulinemia, elevated C-peptide) in this exploratory study. These findings supplement the body of evidence from completed or ongoing OLZ/SAM clinical trials supporting its role in the treatment of schizophrenia and bipolar I disorder., (© 2021. The Author(s).)

Though all theories for the origin of life require a source of energy to promote primordial chemical reactions, the nature of energy that drove the emergence of metabolism at origins is still debated. We reasoned that evidence for the nature of energy at origins should be preserved in the biochemical reactions of life itself, whereby changes in free energy, Δ G , which determine whether a reaction can go forward or not, should help specify the source. By calculating values of Δ G across the conserved and universal core of 402 individual reactions that synthesize amino acids, nucleotides and cofactors from H 2 , CO 2 , NH 3 , H 2 S and phosphate in modern cells, we find that 95-97% of these reactions are exergonic (Δ G ≤ 0 kJ⋅mol -1 ) at pH 7-10 and 80-100°C under nonequilibrium conditions with H 2 replacing biochemical reductants. While 23% of the core's reactions involve ATP hydrolysis, 77% are ATP-independent, thermodynamically driven by Δ G of reactions involving carbon bonds. We identified 174 reactions that are exergonic by -20 to -300 kJ⋅mol -1 at pH 9 and 80°C and that fall into ten reaction types: six pterin dependent alkyl or acyl transfers, ten S-adenosylmethionine dependent alkyl transfers, four acyl phosphate hydrolyses, 14 thioester hydrolyses, 30 decarboxylations, 35 ring closure reactions, 31 aromatic ring formations, and 44 carbon reductions by reduced nicotinamide, flavins, ferredoxin, or formate. The 402 reactions of the biosynthetic core trace to the last universal common ancestor (LUCA), and reveal that synthesis of LUCA's chemical constituents required no external energy inputs such as electric discharge, UV-light or phosphide minerals. The biosynthetic reactions of LUCA uncover a natural thermodynamic tendency of metabolism to unfold from energy released by reactions of H 2 , CO 2 , NH 3 , H 2 S, and phosphate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wimmer, Xavier, Vieira, Pereira, Leidner, Sousa, Kleinermanns, Preiner and Martin.)

The possible evolutionary significance of pyrophosphate (PP i ) has been discussed since the early 1960s. Lipmann suggested that PP i could have been an ancient currency or a possible environmental source of metabolic energy at origins, while Kornberg proposed that PP i vectorializes metabolism because ubiquitous pyrophosphatases render PP i forming reactions kinetically irreversible. To test those ideas, we investigated the reactions that consume phosphoanhydride bonds among the 402 reactions of the universal biosynthetic core that generates amino acids, nucleotides, and cofactors from H 2 , CO 2 , and NH 3 . We find that 36% of the core's phosphoanhydride hydrolyzing reactions generate PP i , while no reactions use PP i as an energy currency. The polymerization reactions that generate ~80% of cell mass - protein, RNA, and DNA synthesis - all generate PP i , while none use PP i as an energy source. In typical prokaryotic cells, aminoacyl tRNA synthetases (AARS) underlie ~80% of PP i production. We show that the irreversibility of the AARS reaction is a kinetic, not a thermodynamic effect. The data indicate that PP i is not an ancient energy currency and probably never was. Instead, PP i hydrolysis is an ancient mechanism that imparts irreversibility, as Kornberg suggested, functioning like a ratchet's pawl to vectorialize the life process toward growth. The two anhydride bonds in nucleoside triphosphates offer ATP-cleaving enzymes an option to impart either thermodynamic control (P i formation) or kinetic control (PP i formation) upon reactions. This dual capacity explains why nature chose the triphosphate moiety of ATP as biochemistry's universal energy currency., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wimmer, Kleinermanns and Martin.)

The contribution of gene duplications to the evolution of eukaryotic genomes is well studied. By contrast, studies of gene duplications in prokaryotes are scarce and generally limited to a handful of genes or careful analysis of a few prokaryotic lineages. Systematic broad-scale studies of prokaryotic genomes that sample available data are lacking, leaving gaps in our understanding of the contribution of gene duplications as a source of genetic novelty in the prokaryotic world. Here, we report conservative and robust estimates for the frequency of recent gene duplications within prokaryotic genomes relative to recent lateral gene transfer (LGT), as mechanisms to generate multiple copies of related sequences in the same genome. We obtain our estimates by focusing on evolutionarily recent events among 5,655 prokaryotic genomes, thereby avoiding vagaries of deep phylogenetic inference and confounding effects of ancient events and differential loss. We find that recent, genome-specific gene duplications are at least 50 times less frequent and probably 100 times less frequent than recent, genome-specific, gene acquisitions via LGT. The frequency of gene duplications varies across lineages and functional categories. The findings improve our understanding of genome evolution in prokaryotes and have far-reaching implications for evolutionary models that entail LGT to gene duplications ratio as a parameter., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Bacteria near-universally contain a cell wall sacculus of murein (peptidoglycan), the synthesis of which has been intensively studied for over 50 years. In striking contrast, archaeal species possess a variety of other cell wall types, none of them closely resembling murein. Interestingly though, one type of archaeal cell wall termed pseudomurein found in the methanogen orders Methanobacteriales and Methanopyrales is a structural analogue of murein in that it contains a glycan backbone that is cross-linked by a L-amino acid peptide. Here, we present taxonomic distribution, gene cluster and phylogenetic analyses that confirm orthologues of 13 bacterial murein biosynthesis enzymes in pseudomurein-containing methanogens, most of which are distantly related to their bacterial counterparts. We also present the first structure of an archaeal pseudomurein peptide ligase from Methanothermus fervidus DSM1088 (Mfer336) to a resolution of 2.5 Å and show that it possesses a similar overall tertiary three domain structure to bacterial MurC and MurD type murein peptide ligases. Taken together the data strongly indicate that murein and pseudomurein biosynthetic pathways share a common evolutionary history., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Modern accounts of eukaryogenesis entail an endosymbiotic encounter between an archaeal host and a proteobacterial endosymbiont, with subsequent evolution giving rise to a unicell possessing a single nucleus and mitochondria. The mononucleate state of the last eukaryotic common ancestor (LECA) is seldom, if ever, questioned, even though cells harboring multiple (syncytia, coenocytes, and polykaryons) are surprisingly common across eukaryotic supergroups. Here, we present a survey of multinucleated forms. Ancestral character state reconstruction for representatives of 106 eukaryotic taxa using 16 different possible roots and supergroup sister relationships, indicate that LECA, in addition to being mitochondriate, sexual, and meiotic, was multinucleate. LECA exhibited closed mitosis, which is the rule for modern syncytial forms, shedding light on the mechanics of its chromosome segregation. A simple mathematical model shows that within LECA's multinucleate cytosol, relationships among mitochondria and nuclei were neither one-to-one, nor one-to-many, but many-to-many, placing mitonuclear interactions and cytonuclear compatibility at the evolutionary base of eukaryotic cell origin. Within a syncytium, individual nuclei and individual mitochondria function as the initial lower-level evolutionary units of selection, as opposed to individual cells, during eukaryogenesis. Nuclei within a syncytium rescue each other's lethal mutations, thereby postponing selection for viable nuclei and cytonuclear compatibility to the generation of spores, buffering transitional bottlenecks at eukaryogenesis. The prokaryote-to-eukaryote transition is traditionally thought to have left no intermediates, yet if eukaryogenesis proceeded via a syncytial common ancestor, intermediate forms have persisted to the present throughout the eukaryotic tree as syncytia but have so far gone unrecognized., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here, we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA's bacterial-derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal-derived and eukaryote-specific genes. The surplus of bacterial-derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host's chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage-specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggests that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Bacteria are the most abundant cells on Earth. They are generally regarded as ancient, but due to striking diversity in their metabolic capacities and widespread lateral gene transfer, the physiology of the first bacteria is unknown. From 1089 reference genomes of bacterial anaerobes, we identified 146 protein families that trace to the last bacterial common ancestor, LBCA, and form the conserved predicted core of its metabolic network, which requires only nine genes to encompass all universal metabolites. Our results indicate that LBCA performed gluconeogenesis towards cell wall synthesis, and had numerous RNA modifications and multifunctional enzymes that permitted life with low gene content. In accordance with recent findings for LUCA and LACA, analyses of thousands of individual gene trees indicate that LBCA was rod-shaped and the first lineage to diverge from the ancestral bacterial stem was most similar to modern Clostridia, followed by other autotrophs that harbor the acetyl-CoA pathway.

The metabolism of cells contains evidence reflecting the process by which they arose. Here, we have identified the ancient core of autotrophic metabolism encompassing 404 reactions that comprise the reaction network from H 2 , CO 2 , and ammonia (NH 3 ) to amino acids, nucleic acid monomers, and the 19 cofactors required for their synthesis. Water is the most common reactant in the autotrophic core, indicating that the core arose in an aqueous environment. Seventy-seven core reactions involve the hydrolysis of high-energy phosphate bonds, furthermore suggesting the presence of a non-enzymatic and highly exergonic chemical reaction capable of continuously synthesizing activated phosphate bonds. CO 2 is the most common carbon-containing compound in the core. An abundance of NADH and NADPH-dependent redox reactions in the autotrophic core, the central role of CO 2 , and the circumstance that the core's main products are far more reduced than CO 2 indicate that the core arose in a highly reducing environment. The chemical reactions of the autotrophic core suggest that it arose from H 2 , inorganic carbon, and NH 3 in an aqueous environment marked by highly reducing and continuously far from equilibrium conditions. Such conditions are very similar to those found in serpentinizing hydrothermal systems.

Metagenomic studies permit the exploration of microbial diversity in a defined habitat, and binning procedures enable phylogenomic analyses, taxon description, and even phenotypic characterizations in the absence of morphological evidence. Such lineages include asgard archaea, which were initially reported to represent archaea with eukaryotic cell complexity, although the first images of such an archaeon show simple cells with prokaryotic characteristics. However, these metagenome-assembled genomes (MAGs) might suffer from data quality problems not encountered in sequences from cultured organisms due to two common analytical procedures of bioinformatics: assembly of metagenomic sequences and binning of assembled sequences on the basis of innate sequence properties and abundance across samples. Consequently, genomic sequences of distantly related taxa, or domains, can in principle be assigned to the same MAG and result in chimeric sequences. The impacts of low-quality or chimeric MAGs on phylogenomic and metabolic prediction remain unknown. Debates that asgard archaeal data are contaminated with eukaryotic sequences are overshadowed by the lack of evidence indicating that individual asgard MAGs stem from the same chromosome. Here, we show that universal proteins including ribosomal proteins of asgard archaeal MAGs fail to meet the basic phylogenetic criterion fulfilled by genome sequences of cultured archaea investigated to date: These proteins do not share common evolutionary histories to the same extent as pure culture genomes do, pointing to a chimeric nature of asgard archaeal MAGs. Our analysis suggests that some asgard archaeal MAGs represent unnatural constructs, genome-like patchworks of genes resulting from assembly and/or the binning process., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In 1910, the Russian biologist Konstantin Sergejewitch Mereschkowsky (Константин Сергеевич Мережковский, in standard transliterations also written as Konstantin Sergeevič Merežkovskij and Konstantin Sergeevich Merezhkovsky) published a notable synthesis of observations and inferences concerning the origin of life and the origin of nucleated cells. His theory was based on physiology and leaned heavily upon the premise that thermophilic autotrophs were ancient. The ancestors of plants and animals were inferred as ancestrally mesophilic anucleate heterotrophs (Monera) that became complex and diverse through endosymbiosis. He placed a phylogenetic root in the tree of life among anaerobic autotrophic bacteria that lack chlorophyll. His higher level classification of all microbes and macrobes in the living world was based upon the presence or absence of past endosymbiotic events. The paper's primary aim was to demonstrate that all life forms descend from two fundamentally distinct organismal lineages, called mykoplasma and amoeboplasma, whose very nature was so different that, in his view, they could only have arisen independently of one another and at different times during Earth history. The mykoplasma arose at a time when the young Earth was still hot, it later gave rise to cyanobacteria, which in turn gave rise to plastids. The product of the second origin of life, the amoeboplasma, arose after the Earth had cooled and autotrophs had generated substrates for heterotrophic growth. Lineage diversification of that second plasma brought forth, via serial endosymbioses, animals (one symbiosis) and then plants (two symbioses, the second being the plastid). The paper was published in German, rendering it inaccessible to many interested scholars. Here we translate the 1910 paper in full and briefly provide some context., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Lateral gene transfer (LGT) has impacted prokaryotic genome evolution, yet the extent to which LGT compromises vertical evolution across individual genes and individual phyla is unknown, as are the factors that govern LGT frequency across genes. Estimating LGT frequency from tree comparisons is problematic when thousands of genomes are compared, because LGT becomes difficult to distinguish from phylogenetic artefacts. Here we report quantitative estimates for verticality across all genes and genomes, leveraging a well-known property of phylogenetic inference: phylogeny works best at the tips of trees. From terminal (tip) phylum level relationships, we calculate the verticality for 19,050,992 genes from 101,422 clusters in 5,655 prokaryotic genomes and rank them by their verticality. Among functional classes, translation, followed by nucleotide and cofactor biosynthesis, and DNA replication and repair are the most vertical. The most vertically evolving lineages are those rich in ecological specialists such as Acidithiobacilli, Chlamydiae, Chlorobi and Methanococcales. Lineages most affected by LGT are the α-, β-, γ-, and δ- classes of Proteobacteria and the Firmicutes. The 2,587 eukaryotic clusters in our sample having prokaryotic homologues fail to reject eukaryotic monophyly using the likelihood ratio test. The low verticality of α-proteobacterial and cyanobacterial genomes requires only three partners-an archaeal host, a mitochondrial symbiont, and a plastid ancestor-each with mosaic chromosomes, to directly account for the prokaryotic origin of eukaryotic genes. In terms of phylogeny, the 100 most vertically evolving prokaryotic genes are neither representative nor predictive for the remaining 97% of an average genome. In search of factors that govern LGT frequency, we find a simple but natural principle: Verticality correlates strongly with gene distribution density, LGT being least likely for intruding genes that must replace a preexisting homologue in recipient chromosomes. LGT is most likely for novel genetic material, intruding genes that encounter no competing copy., Competing Interests: The authors have declared that no competing interests exist.