Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodeling superfamily.

Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.

The cell envelope of Thermotogae suggests a mechanism for outer membrane biogenesis.

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the in situ cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of ß-barrel trimers supporting small (~200 nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompα and novel ß-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions.

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis.

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

On the Nature of the Last Common Ancestor: A Story from its Translation Machinery.

The Last Common Ancestor (LCA) is understood as a hypothetical population of organisms from which all extant living creatures are thought to have descended. Its biology and environment have been and continue to be the subject of discussions within the scientific community. Since the first bacterial genomes were obtained, multiple attempts to reconstruct the genetic content of the LCA have been made. In this review, we compare 10 of the most extensive reconstructions of the gene content possessed by the LCA as they relate to aspects of the translation machinery. Although each reconstruction has its own methodological biases and many disagree in the metabolic nature of the LCA all, to some extent, indicate that several components of the translation machinery are among the most conserved genetic elements. The datasets from each reconstruction clearly show that the LCA already had a largely complete translational system with a genetic code already in place and therefore was not a progenote. Among these features several ribosomal proteins, transcription factors like IF2, EF-G, and EF-Tu and both class I and class II aminoacyl tRNA synthetases were found in essentially all reconstructions. Due to the limitations of the various methodologies, some features such as the occurrence of rRNA posttranscriptional modified bases are not fully addressed. However, conserved as it is, non-universal ribosomal features found in various reconstructions indicate that LCA's translation machinery was still evolving, thereby acquiring the domain specific features in the process. Although progenotes from the pre-LCA likely no longer exist recent results obtained by unraveling the early history of the ribosome and other genetic processes can provide insight to the nature of the pre-LCA world.

Stem Life: A Framework for Understanding the Prebiotic-Biotic Transition.

Abiogenesis is frequently envisioned as a linear, ladder-like progression of increasingly complex chemical systems, eventually leading to the ancestors of extant cellular life. This "pre-cladistics" view is in stark contrast to the well-accepted principles of organismal evolutionary biology, as informed by paleontology and phylogenetics. Applying this perspective to origins, I explore the paradigm of "Stem Life," which embeds abiogenesis within a broader continuity of diversification and extinction of both hereditary lineages and chemical systems. In this new paradigm, extant life's ancestral lineage emerged alongside and was dependent upon many other complex prebiotic chemical systems, as part of a diverse and fecund prebiosphere. Drawing from several natural history analogies, I show how this shift in perspective enriches our understanding of Origins and directly informs debates on defining Life, the emergence of the Last Universal Common Ancestor (LUCA), and the implications of prebiotic chemical experiments.

The Last Universal Common Ancestor of Ribosome-Encoding Organisms: Portrait of LUCA.

The existence of LUCA in the distant past is the logical consequence of the binary mechanism of cell division. The biosphere in which LUCA and contemporaries were living was the product of a long cellular evolution from the origin of life to the second age of the RNA world. A parsimonious scenario suggests that the molecular fabric of LUCA was much simpler than those of modern organisms, explaining why the evolutionary tempo was faster at the time of LUCA than it was during the diversification of the three domains. Although LUCA was possibly equipped with a RNA genome and most likely lacked an ATP synthase, it was already able to perform basic metabolic functions and to produce efficient proteins. However, the proteome of LUCA and its inferred metabolism remains to be correctly explored by in-depth phylogenomic analyses and updated datasets. LUCA was probably a mesophile or a moderate thermophile since phylogenetic analyses indicate that it lacked reverse gyrase, an enzyme systematically present in all hyperthermophiles. The debate about the position of Eukarya in the tree of life, either sister group to Archaea or descendants of Archaea, has important implications to draw the portrait of LUCA. In the second alternative, one can a priori exclude the presence of specific eukaryotic features in LUCA. In contrast, if Archaea and Eukarya are sister group, some eukaryotic features, such as the spliceosome, might have been present in LUCA and later lost in Archaea and Bacteria. The nature of the LUCA virome is another matter of debate. I suggest here that DNA viruses only originated during the diversification of the three domains from an RNA-based LUCA to explain the odd distribution pattern of DNA viruses in the tree of life.

Volatile Organic Compound Metabolism on Early Earth.

Biogenic volatile organic compounds (VOCs) constitute a significant portion of gas-phase metabolites in modern ecosystems and have unique roles in moderating atmospheric oxidative capacity, solar radiation balance, and aerosol formation. It has been theorized that VOCs may account for observed geological and evolutionary phenomena during the Archaean, but the direct contribution of biology to early non-methane VOC cycling remains unexplored. Here, we provide an assessment of all potential VOCs metabolized by the last universal common ancestor (LUCA). We identify enzyme functions linked to LUCA orthologous protein groups across eight literature sources and estimate the volatility of all associated substrates to identify ancient volatile metabolites. We hone in on volatile metabolites with confirmed modern emissions that exist in conserved metabolic pathways and produce a curated list of the most likely LUCA VOCs. We introduce volatile organic metabolites associated with early life and discuss their potential influence on early carbon cycling and atmospheric chemistry.

Ribosome-associated quality control and CAT tailing.

Translation is the set of mechanisms by which ribosomes decode genetic messages as they synthesize polypeptides of a defined amino acid sequence. While the ribosome has been honed by evolution for high-fidelity translation, errors are inevitable. Aberrant mRNAs, mRNA structure, defective ribosomes, interactions between nascent proteins and the ribosomal exit tunnel, and insufficient cellular resources, including low tRNA levels, can lead to functionally irreversible stalls. Life thus depends on quality control mechanisms that detect, disassemble and recycle stalled translation intermediates. Ribosome-associated Quality Control (RQC) recognizes aberrant ribosome states and targets their potentially toxic polypeptides for degradation. Here we review recent advances in our understanding of RQC in bacteria, fungi, and metazoans. We focus in particular on an unusual modification made to the nascent chain known as a "CAT tail", or Carboxy-terminal Alanine and Threonine tail, and the mechanisms by which ancient RQC proteins catalyze CAT-tail synthesis.

Conserved functions of prion candidates suggest a primeval role of protein self-templating.

Amyloid-based prions have simple structures, a wide phylogenetic distribution, and a plethora of functions in contemporary organisms, suggesting they may be an ancient phenomenon. However, this hypothesis has yet to be addressed with a systematic, computational, and experimental approach. Here we present a framework to help guide future experimental verification of candidate prions with conserved functions to understand their role in the early stages of evolution and potentially in the origins of life. We identified candidate prions in all high-quality proteomes available in UniProt computationally, assessed their phylogenomic distributions, and analyzed candidate-prion functional annotations. Of the 27 980 560 proteins scanned, 228 561 were identified as candidate prions (~0.82%). Among these candidates, there were 84 Gene Ontology (GO) terms conserved across the three domains of life. We found that candidate prions with a possible role in adaptation were particularly well-represented within this group. We discuss unifying features of candidate prions to elucidate the primeval roles of prions and their associated functions. Candidate prions annotated as transcription factors, DNA binding, and kinases are particularly well suited to generating diverse responses to changes in their environment and could allow for adaptation and population expansion into more diverse environments. We hypothesized that a relationship between these functions and candidate prions could be evolutionarily ancient, even if individual prion domains themselves are not evolutionarily conserved. Candidate prions annotated with these universally occurring functions potentially represent the oldest extant prions on Earth and are therefore excellent experimental targets.

Investigation of tRNA-based relatedness within the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum: a comparative analysis.

The PVC superphylum is a diverse group of prokaryotes that require stringent growth conditions. RNA is a fascinating molecule to find evolutionary relatedness according to the RNA World Hypothesis. We conducted tRNA gene analysis to find evolutionary relationships in the PVC phyla. The analysis of genomic data (P = 9, V = 4, C = 8) revealed that the number of tRNA genes varied from 28 to 90 in Planctomycetes and Chlamydia, respectively. Verrucomicrobia has whole genomes and the longest scaffold (3 + 1), with tRNA genes ranging from 49 to 53 in whole genomes and 4 in the longest scaffold. Most tRNAs in the E. coli genome clustered with homologs, but approximately 43% clustered with tRNAs encoding different amino acids. Planctomyces, Akkermansia, Isosphaera, and Chlamydia were similar to E. coli tRNAs. In a phylum, tRNAs coding for different amino acids clustered at a range of 8 to 10%. Further analysis of these tRNAs showed sequence similarity with Cyanobacteria, Proteobacteria, Viridiplantae, Ascomycota and Basidiomycota (Eukaryota). This indicates the possibility of horizontal gene transfer or, otherwise, a different origin of tRNA in PVC bacteria. Hence, this work proves its importance for determining evolutionary relatedness and potentially identifying bacteria using tRNA. Thus, the analysis of these tRNAs indicates that primitive RNA may have served as the genetic material of LUCA before being replaced by DNA. A quantitative analysis is required to test these possibilities that relate the evolutionary significance of tRNA to the origin of life.

Evolution of the genetic code; Evidence from serine codon use disparity in Escherichia coli.

Among the 20 amino acids, three of them-leucine (Leu), arginine (Arg), and serine (Ser)-are encoded by six different codons. In comparison, all of the other 17 amino acids are encoded by either 4, 3, 2, or 1 codon. Peculiarly, Ser is separated into two disparate Ser codon boxes, differing by at least two-base substitutions, in contrast to Leu and Arg, of which codons are mutually exchangeable by a single-base substitution. We propose that these two different Ser codons independently emerged during evolution. In this hypothesis, at the time of the origin of life there were only seven primordial amino acids: Valine (coded by GUX [X = U, C, A or G]), alanine (coded by GCX), aspartic acid (coded by GAY [Y = U or C]), glutamic acid (coded by GAZ [Z = A or G]), glycine (coded by GGX), Ser (coded by AGY), and Arg (coded by CGX and AGZ). All of these were derived from GGX for glycine by single-base substitutions. Later in evolution, another class of Ser codons, UCX, were derived from alanine codons, GCX, distinctly different from the other primordial Ser codon, AGY. From the analysis of the Escherichia coli genome, we find extensive disparities in the usage of these two Ser codons, as some genes use only AGY for Ser in their genes. In contrast, others use only UCX, pointing to distinct differences in their origins, consistent with our hypothesis.

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements.

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions-thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.

Coevolutionary and Phylogenetic Analysis of Mimiviral Replication Machinery Suggest the Cellular Origin of Mimiviruses.

Mimivirus is one of the most complex and largest viruses known. The origin and evolution of Mimivirus and other giant viruses have been a subject of intense study in the last two decades. The two prevailing hypotheses on the origin of Mimivirus and other viruses are the reduction hypothesis, which posits that viruses emerged from modern unicellular organisms; whereas the virus-first hypothesis proposes viruses as relics of precellular forms of life. In this study, to gain insights into the origin of Mimivirus, we have carried out extensive phylogenetic, correlation, and multidimensional scaling analyses of the putative proteins involved in the replication of its 1.2-Mb large genome. Correlation analysis and multidimensional scaling methods were validated using bacteriophage, bacteria, archaea, and eukaryotic replication proteins before applying to Mimivirus. We show that a large fraction of mimiviral replication proteins, including polymerase B, clamp, and clamp loaders are of eukaryotic origin and are coevolving. Although phylogenetic analysis places some components along the lineages of phage and bacteria, we show that all the replication-related genes have been homogenized and are under purifying selection. Collectively our analysis supports the idea that Mimivirus originated from a complex cellular ancestor. We hypothesize that Mimivirus has largely retained complex replication machinery reminiscent of its progenitor while losing most of the other genes related to processes such as metabolism and translation.

Evolutionary Origins of Two-Barrel RNA Polymerases and Site-Specific Transcription Initiation.

Evolution-related multisubunit RNA polymerases (RNAPs) carry out RNA synthesis in all domains life. Although their catalytic cores and fundamental mechanisms of transcription elongation are conserved, the initiation stage of the transcription cycle differs substantially in bacteria, archaea, and eukaryotes in terms of the requirements for accessory factors and details of the molecular mechanisms. This review focuses on recent insights into the evolution of the transcription apparatus with regard to (a) the surprisingly pervasive double-Ψ ß-barrel active-site configuration among different nucleic acid polymerase families, (b) the origin and phylogenetic distribution of TBP, TFB, and TFE transcription factors, and

Fine tuned exploration of evolutionary relationships within the protein universe.

In the regime of domain classifications, the protein universe unveils a discrete set of folds connected by hierarchical relationships. Instead, at sub-domain-size resolution and because of physical constraints not necessarily requiring evolution to shape polypeptide chains, networks of protein motifs depict a continuous view that lies beyond the extent of hierarchical classification schemes. A number of studies, however, suggest that universal sub-sequences could be the descendants of peptides emerged in an ancient pre-biotic world. Should this be the case, evolutionary signals retained by structurally conserved motifs, along with hierarchical features of ancient domains, could sew relationships among folds that diverged beyond the point where homology is discernable. In view of the aforementioned, this paper provides a rationale where a network with hierarchical and continuous levels of the protein space, together with sequence profiles that probe the extent of sequence similarity and contacting residues that capture the transition from pre-biotic to domain world, has been used to explore relationships between ancient folds. Statistics of detected signals have been reported. As a result, an example of an emergent sub-network that makes sense from an evolutionary perspective, where conserved signals retrieved from the assessed protein space have been co-opted, has been discussed.

A New Analysis of Archaea-Bacteria Domain Separation: Variable Phylogenetic Distance and the Tempo of Early Evolution.

Comparative genomics and molecular phylogenetics are foundational for understanding biological evolution. Although many studies have been made with the aim of understanding the genomic contents of early life, uncertainty remains. A study by Weiss et al. (Weiss MC, Sousa FL, Mrnjavac N, Neukirchen S, Roettger M, Nelson-Sathi S, Martin WF. 2016. The physiology and habitat of the last universal common ancestor. Nat Microbiol. 1(9):16116.) identified a number of protein families in the last universal common ancestor of archaea and bacteria (LUCA) which were not found in previous works. Here, we report new research that suggests the clustering approaches used in this previous study undersampled protein families, resulting in incomplete phylogenetic trees which do not reflect protein family evolution. Phylogenetic analysis of protein families which include more sequence homologs rejects a simple LUCA hypothesis based on phylogenetic separation of the bacterial and archaeal domains for a majority of the previously identified LUCA proteins (∼82%). To supplement limitations of phylogenetic inference derived from incompletely populated orthologous groups and to test the hypothesis of a period of rapid evolution preceding the separation of the domains, we compared phylogenetic distances both within and between domains, for thousands of orthologous groups. We find a substantial diversity of interdomain versus intradomain branch lengths, even among protein families which exhibit a single domain separating branch and are thought to be associated with the LUCA. Additionally, phylogenetic trees with long interdomain branches relative to intradomain branches are enriched in information categories of protein families in comparison to those associated with metabolic functions. These results provide a new view of protein family evolution and temper claims about the phenotype and habitat of the LUCA.

The Origin(s) of Cell(s): Pre-Darwinian Evolution from FUCAs to LUCA : To Carl Woese (1928-2012), for his Conceptual Breakthrough of Cellular Evolution.

The coming of the Last Universal Cellular Ancestor (LUCA) was the singular watershed event in the making of the biotic world. If the coming of LUCA marked the crossing of the "Darwinian Threshold", then pre-LUCA evolution must have been Pre-Darwinian and at least partly non-Darwinian. But how did Pre-Darwinian evolution before LUCA actually operate? I broaden our understanding of the central mechanism of biological evolution (i.e., variation-selection-inheritance) and then extend this broadened understanding to its natural starting point: the origin(s) of the First Universal Cellular Ancestors (FUCAs) before LUCA. My hypothesis centers upon vesicles' making-and-remaking as variation and competition as selection. More specifically, I argue that vesicles' acquisition and merger, via breaking-and-repacking, proto-endocytosis, proto-endosymbiosis, and other similar processes had been a central force of both variation and selection in the pre-Darwinian epoch. These new perspectives shed important new light upon the origin of FUCAs and their subsequent evolution into LUCA.

Protein-free ribosomal RNA scaffolds can assemble poly-lysine oligos from charged tRNA fragments.

Ribosomal protein synthesis is a central process of the modern biological world. Because the ribosome contains proteins itself, it is very important to understand its precursor and evolution. Small ribozymes have demonstrated the principle of "RNA world" hypothesis, but protein free peptide ligase remains elusive. In this report, we have identified two fragments in the peptidyl transfer center that can synthesize a 9-mer poly-lysine in a solution contains Mg2+. This result is deduced from isotope-shifting in high resolution MS. To our best knowledge, this is the longest peptide oligo that can be synthesized by a pure ribozyme. Via single molecule FRET experiments, we have demonstrated the ligase mechanism was probably by substrate proximity via dimerization. We prospect that these RNA fragments can be useful to synthesize template free natural and non-natural peptides, to be model system for peptidyl transfer reaction mechanism and can shed light to the evolution of ribosome.

Supraventricular tachycardias in neonates and infants: factors associated with fatal or near-fatal outcome.

Prognosis of supraventricular tachycardias in neonates and infants is thought to be excellent with rare fatal outcomes. Nevertheless, initial management can be challenging. The aim of this study was to perform a retrospective analysis in neonates/infants with non-pos-toperative supraventricular tachycardias regarding risk factors for clinical outcome and type of antiarrhythmic drug therapy. The data of 157 patients aged < 1 year who presented between 2000 and 2015 with symptomatic tachycardias were retrospectively reviewed. Pharmacological therapy was successful in 151 patients (96%); 1 patient (1%) required catheter ablation and 5 patients (3%) died (1 death linked to hemodynamical reasons after effective arrhythmia control). Serious complications following acute medical therapy occurred in 4 patients of survivors. Patients with complications or death had a lower bodyweight, more frequent intrauterine tachycardia, transplacental therapy, urgent caesarian section, higher PRISM II score, longer period to control tachycardia, more frequent proarrhythmia, and major adverse event-defined as life-threatening event without a documented new arrhythmia-compared to the group without complications. There was no significant difference between the groups regarding prematurity, structural heart disease, and type of tachycardia. Proarrhythmia occurred in 6 cases and was related to intravenous drug use with class IC antiarrhythmics in 3/6 cases, digoxin in 2/6 cases, and amiodarone in 1/6 cases. ECG signs of impending proarrhythmia without new-onset arrhythmia requiring cessation of therapy were detected in 6 patients.Conclusion: Although rare, non-post-operative supraventricular tachycardia in neonates and infants might be a serious disease. Acute intravenous pharmacological treatment to control tachycardia might pose a risk for fatal or near-fatal outcome. Detection of proarrhythmia related to class IC antiarrhythmics in neonates might be especially difficult and requires alertness. What is Known • Prognosis of supraventricular tachycardias in children are thought to be excellent with fatal outcomes being rare. • Mortality is increased in the very young and in those with structural heart disease. What is New • Complicated outcome of non-post-operative supraventricular tachycardias in neonates is associated with lower bodyweight, age, prenatal tachycardia, higher PRISM II score, longer period to control tachycardia, and proarrhythmia. • Detection of class IC proarrhythmic effect is especially difficult in neonates because of their narrow QRS and warrants alertness.

Why the Lipid Divide? Membrane Proteins as Drivers of the Split between the Lipids of the Three Domains of Life.

Recent results from engineered and natural samples show that the starkly different lipids of archaea and bacteria can form stable hybrid membranes. But if the two types can mix, why don't they? That is, why do most bacteria and all eukaryotes have only typically bacterial lipids, and archaea archaeal lipids? It is suggested here that the reason may lie on the other main component of cellular membranes: membrane proteins, and their close adaptation to the lipids. Archaeal lipids in modern bacteria could suggest that the last universal common ancestor (LUCA) had both lipid types. However, this would imply a rather elaborate evolutionary scenario, while negating simpler alternatives. In light of widespread horizontal gene transfer across the prokaryotic domains, hybrid membranes reveal that the lipid divide did not just occur once at the divergence of archaea and bacteria from LUCA. Instead, it continues to occur actively to this day.