Paramecium Genetics, Genomics, and Evolution.

The ciliate genus Paramecium served as one of the first model systems in microbial eukaryotic genetics, contributing much to the early understanding of phenomena as diverse as genome rearrangement, cryptic speciation, cytoplasmic inheritance, and endosymbiosis, as well as more recently to the evolution of mating types, introns, and roles of small RNAs in DNA processing. Substantial progress has recently been made in the area of comparative and population genomics. Paramecium species combine some of the lowest known mutation rates with some of the largest known effective populations, along with likely very high recombination rates, thereby harboring a population-genetic environment that promotes an exceptionally efficient capacity for selection. As a consequence, the genomes are extraordinarily streamlined, with very small intergenic regions combined with small numbers of tiny introns. The subject of the bulk of Paramecium research, the ancient Paramecium aurelia species complex, is descended from two whole-genome duplication events that retain high degrees of synteny, thereby providing an exceptional platform for studying the fates of duplicate genes. Despite having a common ancestor dating to several hundred million years ago, the known descendant species are morphologically indistinguishable, raising significant questions about the common view that gene duplications lead to the origins of evolutionary novelties.

The genome-wide signature of short-term temporal selection.

Despite evolutionary biology's obsession with natural selection, few studies have evaluated multigenerational series of patterns of selection on a genome-wide scale in natural populations. Here, we report on a 10-y population-genomic survey of the microcrustacean Daphnia pulex. The genome sequences of [Formula: see text]800 isolates provide insights into patterns of selection that cannot be obtained from long-term molecular-evolution studies, including the following: the pervasiveness of near quasi-neutrality across the genome (mean net selection coefficients near zero, but with significant temporal variance about the mean, and little evidence of positive covariance of selection across time intervals); the preponderance of weak positive selection operating on minor alleles; and a genome-wide distribution of numerous small linkage islands of observable selection influencing levels of nucleotide diversity. These results suggest that interannual fluctuating selection is a major determinant of standing levels of variation in natural populations, challenge the conventional paradigm for interpreting patterns of nucleotide diversity and divergence, and motivate the need for the further development of theoretical expressions for the interpretation of population-genomic data.

Mutation pressure, drift, and the pace of molecular coevolution.

Most aspects of the molecular biology of cells involve tightly coordinated intermolecular interactions requiring specific recognition at the nucleotide and/or amino acid levels. This has led to long-standing interest in the degree to which constraints on interacting molecules result in conserved vs. accelerated rates of sequence evolution, with arguments commonly being made that molecular coevolution can proceed at rates exceeding the neutral expectation. Here, a fairly general model is introduced to evaluate the degree to which the rate of evolution at functionally interacting sites is influenced by effective population sizes (Ne), mutation rates, strength of selection, and the magnitude of recombination between sites. This theory is of particular relevance to matters associated with interactions between organelle- and nuclear-encoded proteins, as the two genomic environments often exhibit dramatic differences in the power of mutation and drift. Although genes within low Ne environments can drive the rate of evolution of partner genes experiencing higher Ne, rates exceeding the neutral expectation require that the former also have an elevated mutation rate. Testable predictions, some counterintuitive, are presented on how patterns of coevolutionary rates should depend on the relative intensities of drift, selection, and mutation.

The fidelity of transcription in human cells.

To determine the error rate of transcription in human cells, we analyzed the transcriptome of H1 human embryonic stem cells with a circle-sequencing approach that allows for high-fidelity sequencing of the transcriptome. These experiments identified approximately 100,000 errors distributed over every major RNA species in human cells. Our results indicate that different RNA species display different error rates, suggesting that human cells prioritize the fidelity of some RNAs over others. Cross-referencing the errors that we detected with various genetic and epigenetic features of the human genome revealed that the in vivo error rate in human cells changes along the length of a transcript and is further modified by genetic context, repetitive elements, epigenetic markers, and the speed of transcription. Our experiments further suggest that BRCA1, a DNA repair protein implicated in breast cancer, has a previously unknown role in the suppression of transcription errors. Finally, we analyzed the distribution of transcription errors in multiple tissues of a new mouse model and found that they occur preferentially in neurons, compared to other cell types. These observations lend additional weight to the idea that transcription errors play a key role in the progression of various neurological disorders, including Alzheimer's disease.

Both Las17-binding sites on Arp2/3 complex are important for branching nucleation and assembly of functional endocytic actin networks in S. cerevisiae.

Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site.

A chemosensory-like histidine kinase is dispensable for chemotaxis in vitro but regulates the virulence of Borrelia burgdorferi through modulating the stability of RpoS.

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1ß, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.

Recommendations for improving statistical inference in population genomics.

The field of population genomics has grown rapidly in response to the recent advent of affordable, large-scale sequencing technologies. As opposed to the situation during the majority of the 20th century, in which the development of theoretical and statistical population genetic insights outpaced the generation of data to which they could be applied, genomic data are now being produced at a far greater rate than they can be meaningfully analyzed and interpreted. With this wealth of data has come a tendency to focus on fitting specific (and often rather idiosyncratic) models to data, at the expense of a careful exploration of the range of possible underlying evolutionary processes. For example, the approach of directly investigating models of adaptive evolution in each newly sequenced population or species often neglects the fact that a thorough characterization of ubiquitous nonadaptive processes is a prerequisite for accurate inference. We here describe the perils of these tendencies, present our consensus views on current best practices in population genomic data analysis, and highlight areas of statistical inference and theory that are in need of further attention. Thereby, we argue for the importance of defining a biologically relevant baseline model tuned to the details of each new analysis, of skepticism and scrutiny in interpreting model fitting results, and of carefully defining addressable hypotheses and underlying uncertainties.

The divergence of mutation rates and spectra across the Tree of Life.

Owing to advances in genome sequencing, genome stability has become one of the most scrutinized cellular traits across the Tree of Life. Despite its centrality to all things biological, the mutation rate (per nucleotide site per generation) ranges over three orders of magnitude among species and several-fold within individual phylogenetic lineages. Within all major organismal groups, mutation rates scale negatively with the effective population size of a species and with the amount of functional DNA in the genome. This relationship is most parsimoniously explained by the drift-barrier hypothesis, which postulates that natural selection typically operates to reduce mutation rates until further improvement is thwarted by the power of random genetic drift. Despite this constraint, the molecular mechanisms underlying DNA replication fidelity and repair are free to wander, provided the performance of the entire system is maintained at the prevailing level. The evolutionary flexibility of the mutation rate bears on the resolution of several prior conundrums in phylogenetic and population-genetic analysis and raises challenges for future applications in these areas.

Evolutionary Insights from a Large-Scale Survey of Population-Genomic Variation.

The field of genomics has ushered in new methods for studying molecular-genetic variation in natural populations. However, most population-genomic studies still rely on small sample sizes (typically, <100 individuals) from single time points, leaving considerable uncertainties with respect to the behavior of relatively young (and rare) alleles and, owing to the large sampling variance of measures of variation, to the specific gene targets of unusually strong selection. Genomic sequences of ∼1,700 haplotypes distributed over a 10-year period from a natural population of the microcrustacean Daphnia pulex reveal evolutionary-genomic features at a refined scale, including previously hidden information on the behavior of rare alleles predicted by recent theory. Background selection, resulting from the recurrent introduction of deleterious alleles, appears to strongly influence the dynamics of neutral alleles, inducing indirect negative selection on rare variants and positive selection on common variants. Temporally fluctuating selection increases the persistence of nonsynonymous alleles with intermediate frequencies, while reducing standing levels of variation at linked silent sites. Combined with the results from an equally large metapopulation survey of the study species, classes of genes that are under strong positive selection can now be confidently identified in this key model organism. Most notable among rapidly evolving Daphnia genes are those associated with ribosomes, mitochondrial functions, sensory systems, and lifespan determination.

Dynamics of Gene Loss following Ancient Whole-Genome Duplication in the Cryptic Paramecium Complex.

Whole-genome duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods, and the relative contributions of different selective pressures to their maintenance are still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the Paramecium aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and 1 additional out group, revealing aspects of post-WGD evolution in 13 species sharing a common ancestral WGD. Contrary to the morphological radiation of vertebrates that putatively followed two WGD events, members of the cryptic P. aurelia complex have remained morphologically indistinguishable after hundreds of millions of years. Biases in gene retention compatible with dosage constraints appear to play a major role opposing post-WGD gene loss across all 13 species. In addition, post-WGD gene loss has been slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. A near complete lack of recent single-gene duplications in Paramecium provides additional evidence for strong selective pressures against gene dosage changes. This exceptional data set of 13 species sharing an ancestral WGD and 2 closely related out group species will be a useful resource for future studies on Paramecium as a major model organism in the evolutionary cell biology.

Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

The use of buprenorphine to-go packs in the emergency department.

OBJECTIVE: Buprenorphine is an effective treatment for opioid use disorder (OUD). Patients in the emergency department (ED) can be initiated or continued on buprenorphine as a bridge to follow-up in the outpatient setting, but gaps in care may arise. The objective was to evaluate the impact of buprenorphine to-go packs as a continuing treatment option for patients presenting to the ED with OUD across a health system. METHODS: Adult patients discharged with a buprenorphine to-go pack from one of ten EDs within a major health system were included. The primary outcomes assessed within 30 days of ED discharge were: (1) return to a health system ED, and (2) fill history of buprenorphine in the state prescription drug monitoring program database. Data was analyzed using descriptive statistics in Microsoft Excel (Redmond, WA). RESULTS: A total of 124 patients received buprenorphine to-go packs. The sample was primarily male (79; 63.7%), white (89; 71.8%), on Medicaid (79; 63.7%), and had a mean age of 40.9 years. A total of 43 patients (34.7%) were initiated on buprenorphine for the first time, while 81 (65.3%) had received buprenorphine (prescription or to-go) previously. At 30 days post-visit, 76 (61.3%) had filled buprenorphine prescriptions, and 40 (32.3%) returned to an ED within the health system for opioid withdrawal (17; 42.5%), non-OUD-related reasons (22; 55%), or overdose (1; 2.5%). CONCLUSION: The implementation of a system-wide buprenorphine to-go supply at ED discharge is a feasible option to provide continuity of care to patients with OUD.

Physical bioenergetics: Energy fluxes, budgets, and constraints in cells.

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.

Safety and immunogenicity of an adjuvanted recombinant spike protein-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, SpikeVet™, in selected Carnivora, Primates and Artiodactyla in Australian zoos.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad range of animal species and has been associated with severe disease in some taxa. Few studies have evaluated optimal strategies to mitigate the risk to susceptible zoo animals. This study evaluated the safety and immunogenicity of a protein-based veterinary SARS-CoV-2 vaccine (SpikeVet™) in zoo animals. Two to three doses of SpikeVet™ were administered intramuscularly or subcutaneously 3-4 weeks apart to 354 zoo animals representing 38 species. SpikeVet™ was very well tolerated across all species. Minor adverse effects were observed in 1.69% of animals vaccinated, or 1.04% of vaccine doses administered. Preliminary immunogenicity analyses in representative carnivores (meerkats, lions) and an artiodactylid (domestic goat) showed SpikeVet™-immunized animals developed serum antibodies able to neutralize a range of SARS-CoV-2 variants, including the vaccine-homologous Wuhan and Mu variants, as well as vaccine-heterologous Omicron BA.2 and XBB.1 strains. Prior to vaccination, all eight lions were seropositive for Wuhan strain by surrogate viral neutralization testing, suggesting past infection with SARS-CoV-2 or cross-reactive antibodies generated by another closely related coronavirus. These results from a range of zoo species support the ongoing development of SpikeVet™ as a safe and effective veterinary SARS-CoV-2 vaccine.

Pilot examination of independent and interactive effects of maltreatment and neighborhood risk on child attachment.

Elucidating the influence of microsystem and exosystem factors on development is an important goal of developmental psychopathology. This study examined the effects of maltreatment and neighborhood risk on child-caregiver attachment. Maltreatment records, neighborhood risk indices, and Strange Situation data were collected from a diverse sample of 170 four-year-old children and their caregivers. Relative contributions of maltreatment, neighborhood risk, and their interaction on attachment insecurity and disorganization were explored via latent moderation. Maltreated children demonstrated higher rates of insecure attachment, but not attachment disorganization, independent of neighborhood risk. Controlling for maltreatment, preliminary results suggested no effects of neighborhood risk on attachment. Findings support prior research that has identified maltreatment as a salient risk to the formation of secure attachment relationships. However, results add heterogeneity to the limited research investigating effects of neighborhood on attachment. Overall, this study highlights the importance of examining multilevel ecological risk in relation to attachment relationship development.

Evolutionary Genomics of a Subdivided Species.

The ways in which genetic variation is distributed within and among populations is a key determinant of the evolutionary features of a species. However, most comprehensive studies of these features have been restricted to studies of subdivision in settings known to have been driven by local adaptation, leaving our understanding of the natural dispersion of allelic variation less than ideal. Here, we present a geographic population-genomic analysis of 10 populations of the freshwater microcrustacean Daphnia pulex, an emerging model system in evolutionary genomics. These populations exhibit a pattern of moderate isolation-by-distance, with an average migration rate of 0.6 individuals per generation, and average effective population sizes of ∼650,000 individuals. Most populations contain numerous private alleles, and genomic scans highlight the presence of islands of excessively high population subdivision for more common alleles. A large fraction of such islands of population divergence likely reflect historical neutral changes, including rare stochastic migration and hybridization events. The data do point to local adaptive divergence, although the precise nature of the relevant variation is diffuse and cannot be associated with particular loci, despite the very large sample sizes involved in this study. In contrast, an analysis of between-species divergence highlights positive selection operating on a large set of genes with functions nearly nonoverlapping with those involved in local adaptation, in particular ribosome structure, mitochondrial bioenergetics, light reception and response, detoxification, and gene regulation. These results set the stage for using D. pulex as a model for understanding the relationship between molecular and cellular evolution in the context of natural environments.

A Population-Genetic Lens into the Process of Gene Loss Following Whole-Genome Duplication.

Whole-genome duplications (WGDs) have occurred in many eukaryotic lineages. However, the underlying evolutionary forces and molecular mechanisms responsible for the long-term retention of gene duplicates created by WGDs are not well understood. We employ a population-genomic approach to understand the selective forces acting on paralogs and investigate ongoing duplicate-gene loss in multiple species of Paramecium that share an ancient WGD. We show that mutations that abolish protein function are more likely to be segregating in retained WGD paralogs than in single-copy genes, most likely because of ongoing nonfunctionalization post-WGD. This relaxation of purifying selection occurs in only one WGD paralog, accompanied by the gradual fixation of nonsynonymous mutations and reduction in levels of expression, and occurs over a long period of evolutionary time, "marking" one locus for future loss. Concordantly, the fitness effects of new nonsynonymous mutations and frameshift-causing indels are significantly more deleterious in the highly expressed copy compared with their paralogs with lower expression. Our results provide a novel mechanistic model of gene duplicate loss following WGDs, wherein selection acts on the sum of functional activity of both duplicate genes, allowing the two to wander in expression and functional space, until one duplicate locus eventually degenerates enough in functional efficiency or expression that its contribution to total activity is too insignificant to be retained by purifying selection. Retention of duplicates by such mechanisms predicts long times to duplicate-gene loss, which should not be falsely attributed to retention due to gain/change in function.

Genetic Diversity, Heteroplasmy, and Recombination in Mitochondrial Genomes of Daphnia pulex, Daphnia pulicaria, and Daphnia obtusa.

Genetic variants of mitochondrial DNA at the individual (heteroplasmy) and population (polymorphism) levels provide insight into their roles in multiple cellular and evolutionary processes. However, owing to the paucity of genome-wide data at the within-individual and population levels, the broad patterns of these two forms of variation remain poorly understood. Here, we analyze 1,804 complete mitochondrial genome sequences from Daphnia pulex, Daphnia pulicaria, and Daphnia obtusa. Extensive heteroplasmy is observed in D. obtusa, where the high level of intraclonal divergence must have resulted from a biparental-inheritance event, and recombination in the mitochondrial genome is apparent, although perhaps not widespread. Global samples of D. pulex reveal remarkably low mitochondrial effective population sizes, <3% of those for the nuclear genome. In addition, levels of population diversity in mitochondrial and nuclear genomes are uncorrelated across populations, suggesting an idiosyncratic evolutionary history of mitochondria in D. pulex. These population-genetic features appear to be a consequence of background selection associated with highly deleterious mutations arising in the strongly linked mitochondrial genome, which is consistent with polymorphism and divergence data suggesting a predominance of strong purifying selection. Nonetheless, the fixation of mildly deleterious mutations in the mitochondrial genome also appears to be driving positive selection on genes encoded in the nuclear genome whose products are deployed in the mitochondrion.

Rates of Mutations and Transcript Errors in the Foodborne Pathogen Salmonella enterica subsp. enterica.

Because errors at the DNA level power pathogen evolution, a systematic understanding of the rate and molecular spectra of mutations could guide the avoidance and treatment of infectious diseases. We thus accumulated tens of thousands of spontaneous mutations in 768 repeatedly bottlenecked lineages of 18 strains from various geographical sites, temporal spread, and genetic backgrounds. Entailing over ∼1.36 million generations, the resultant data yield an average mutation rate of ∼0.0005 per genome per generation, with a significant within-species variation. This is one of the lowest bacterial mutation rates reported, giving direct support for a high genome stability in this pathogen resulting from high DNA-mismatch-repair efficiency and replication-machinery fidelity. Pathogenicity genes do not exhibit an accelerated mutation rate, and thus, elevated mutation rates may not be the major determinant for the diversification of toxin and secretion systems. Intriguingly, a low error rate at the transcript level is not observed, suggesting distinct fidelity of the replication and transcription machinery. This study urges more attention on the most basic evolutionary processes of even the best-known human pathogens and deepens the understanding of their genome evolution.

Safe administration of Crotalidae equine immune F(ab')2 antivenom in a patient who suffered anaphylaxis from Crotalidae polyvalent immune Fab antivenom.

Allergic reactions to Crotalidae polyvalent immune Fab and Crotalidae immune F(ab')2 are uncommon but potentially life-threatening. It is unknown whether cross-reactivity reactions exist between these two antivenoms. We report a case of a patient who suffered anaphylaxis from Crotalidae polyvalent immune Fab but subsequently was safely administered Crotalidae immune F(ab')2 after a presumed Agkistrodon contortix (copperhead) envenomation. This single case supports the safety of Crotalidae immune F(ab')2 administration in patients with a history of anaphylaxis to Crotalidae polyvalent immune Fab treatment.