Structurally divergent and recurrently mutated regions of primate genomes.

We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.

Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

Characterizing the Major Structural Variant Alleles of the Human Genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.

Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes.

We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.

Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

High resolution genomes of multiple Xiphophorus species provide new insights into microevolution, hybrid incompatibility, and epistasis.

Because of diverged adaptative phenotypes, fish species of the genus Xiphophorus have contributed to a wide range of research for a century. Existing Xiphophorus genome assemblies are not at the chromosomal level and are prone to sequence gaps, thus hindering advancement of the intra- and inter-species differences for evolutionary, comparative, and translational biomedical studies. Herein, we assembled high-quality chromosome-level genome assemblies for three distantly related Xiphophorus species, namely, X. maculatus, X. couchianus, and X. hellerii Our overall goal is to precisely assess microevolutionary processes in the clade to ascertain molecular events that led to the divergence of the Xiphophorus species and to progress understanding of genetic incompatibility to disease. In particular, we measured intra- and inter-species divergence and assessed gene expression dysregulation in reciprocal interspecies hybrids among the three species. We found expanded gene families and positively selected genes associated with live bearing, a special mode of reproduction. We also found positively selected gene families are significantly enriched in nonpolymorphic transposable elements, suggesting the dispersal of these nonpolymorphic transposable elements has accompanied the evolution of the genes, possibly by incorporating new regulatory elements in support of the Britten-Davidson hypothesis. We characterized inter-specific polymorphisms, structural variants, and polymorphic transposable element insertions and assessed their association to interspecies hybridization-induced gene expression dysregulation related to specific disease states in humans.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta.

Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.

Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production.

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

An Early Treatment With BKI-1748 Exhibits Full Protection Against Abortion and Congenital Infection in Sheep Experimentally Infected With Toxoplasma gondii.

Congenital toxoplasmosis in humans and in other mammalian species, such as small ruminants, is a well-known cause of abortion and fetal malformations. The calcium-dependent protein kinase 1 (CDPK1) inhibitor BKI-1748 has shown a promising safety profile for its use in humans and a good efficacy against Toxoplasma gondii infection in vitro and in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg did not show systemic or pregnancy-related toxicity. In sheep experimentally infected at 90 days of pregnancy with 1000 TgShSp1 oocysts, the BKI-1748 treatment administered from 48 hours after infection led to complete protection against abortion and congenital infection. In addition, compared to infected/untreated sheep, treated sheep showed a drastically lower rectal temperature increase and none showed IgG seroconversion throughout the study. In conclusion, BKI-1748 treatment in pregnant sheep starting at 48 hours after infection was fully effective against congenital toxoplasmosis.

Fixation of allelic gene expression landscapes and expression bias pattern shape the transcriptome of the clonal Amazon molly.

The Amazon molly is a unique clonal fish species that originated from an interspecies hybrid between Poecilia species P. mexicana and P. latipinna It reproduces by gynogenesis, which eliminates paternal genomic contribution to offspring. An earlier study showed that Amazon molly shows biallelic expression for a large portion of the genome, leading to two main questions: (1) Are the allelic expression patterns from the initial hybridization event stabilized or changed during establishment of the asexual species and its further evolution? (2) Is allelic expression biased toward one parental allele a stochastic or adaptive process? To answer these questions, the allelic expression of P. formosa siblings was assessed to investigate intra- and inter-cohort allelic expression variability. For comparison, interspecies hybrids between P. mexicana and P. latipinna were produced in the laboratory to represent the P. formosa ancestor. We have identified inter-cohort and intra-cohort variation in parental allelic expression. The existence of inter-cohort divergence suggests functional P. formosa allelic expression patterns do not simply reflect the atavistic situation of the first interspecies hybrid but potentially result from long-term selection of transcriptional fitness. In addition, clonal fish show a transcriptional trend representing minimal intra-clonal variability in allelic expression patterns compared to the corresponding hybrids. The intra-clonal similarity in gene expression translates to sophisticated genetic functional regulation at the individuum level. These findings suggest the parental alleles inherited by P. formosa form tightly regulated genetic networks that lead to a stable transcriptomic landscape within clonal individuals.

Single-cell insights into development of the bovine placenta†.

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation.

Eliminating Qubit-Type Cross-Talk in the omg Protocol.

The omg protocol is a promising paradigm that uses multiple, application-specific, qubit subspaces within the Hilbert space of each single atom during quantum information processing. A key assumption for omg operation is that a subspace can be accessed independently without deleterious effects on information stored in other subspaces. We find that intensity noise during laser-based quantum gates in one subspace can cause decoherence in other subspaces, potentially complicating omg operation. We show, however, that a magnetic-field-induced vector light shift can be used to eliminate this source of decoherence. As this technique simply requires choosing a specific, magnetic field-dependent polarization for the gate lasers, it is straightforward to implement and potentially helpful for omg-based quantum technology.

Intermittent energy restriction inhibits tumor growth and enhances paclitaxel response in a transgenic mouse model of endometrial cancer.

OBJECTIVE: Overweight/obesity is the strongest risk factor for endometrial cancer (EC), and weight management can reduce that risk and improve survival. We aimed to establish the differential benefits of intermittent energy restriction (IER) and low-fat diet (LFD), alone and in combination with paclitaxel, to reverse the procancer effects of high-fat diet (HFD)-induced obesity in a mouse model of EC. METHODS: Lkb1fl/flp53fl/fl mice were fed HFD or LFD to generate obese and lean phenotypes, respectively. Obese mice were maintained on a HFD or switched to a LFD (HFD-LFD) or IER (HFD-IER). Ten weeks after induction of endometrial cancer, mice in each group received paclitaxel or placebo for 4 weeks. Body and tumor weights; tumoral transcriptomic, metabolomic and oxylipin profiles; and serum metabolic hormones and chemocytokines were assessed. RESULTS: HFD-IER and HFD-LFD, relative to HFD, reduced body weight; reversed obesity-induced alterations in serum insulin, leptin and inflammatory factors; and decreased tumor incidence and mass, often to levels emulating those associated with continuous LFD. Concurrent paclitaxel, versus placebo, enhanced tumor suppression in each group, with greatest benefit in HFD-IER. The diets produced distinct tumoral gene expression and metabolic profiles, with HFD-IER associated with a more favorable (antitumor) metabolic and inflammatory environment. CONCLUSION: In Lkb1fl/flp53fl/fl mice, IER is generally more effective than LFD in promoting weight loss, inhibiting obesity-related endometrial tumor growth (particularly in combination with paclitaxel), and reversing detrimental obesity-related metabolic effects. These findings lay the foundation for further investigations of IER as an EC prevention and treatment strategies in overweight/obesity women.

Identifying gene expression programs in single-cell RNA-seq data using linear correlation explanation.

OBJECTIVE: Gene expression analysis through single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of gene regulation in diverse cell types, tissues, and organisms. While existing methods primarily focus on identifying cell type-specific gene expression programs (GEPs), the characterization of GEPs associated with biological processes and stimuli responses remains limited. In this study, we aim to infer biologically meaningful GEPs that are associated with both cellular phenotypes and activity programs directly from scRNA-seq data. METHODS: We applied linear CorEx, a machine-learning-based approach, to infer GEPs by grouping genes based on total correlation optimization function in simulated and real-world scRNA-seq datasets. Additionally, we utilized a transfer learning approach to project CorEx-inferred GEPs to other scRNA-seq datasets. RESULTS: By leveraging total correlation optimization, linear CorEx groups genes and demonstrates superior performance in identifying cell types and activity programs compared to similar methods using simulated data. Furthermore, we apply this same approach to real-world scRNA-seq data from the mouse dentate gyrus and embryonic colon development, uncovering biologically relevant GEPs related to cell types, developmental ages, and cell cycle programs. We also demonstrate the potential for transfer learning by evaluating similar datasets, showcasing the cross-species sensitivity of linear CorEx. CONCLUSION: Our findings validate linear CorEx as a valuable tool for comprehensively analyzing complex signals in scRNA-seq data, leading to deeper insights into gene expression dynamics, cellular heterogeneity, and regulatory mechanisms.

Lion Localizer: A software tool for inferring the provenance of lions (Panthera leo) using mitochondrial DNA.

The illegal poaching of lions for their body parts poses a severe threat to lion populations across Africa. Poaching accounts for 35% of all human-caused lion deaths, with 51% attributed to retaliatory killings following livestock predation. In nearly half of the retaliatory killings, lion body parts are removed, suggesting that high demand for lion body parts may fuel killings attributed to human-lion conflict. Trafficked items are often confiscated in transit or destination countries far from their country of origin. DNA from lion parts may in some cases be the only available means for examining their geographic origins. In this paper, we present the Lion Localizer, a full-stack software tool that houses a comprehensive database of lion mitochondrial DNA (mtDNA) sequences sourced from previously published studies. The database covers 146 localities from across the African continent and India, providing information on the potential provenance of seized lion body parts. Lion mtDNA sequences of 350 or 1,140 bp corresponding to the cytochrome b region can be generated from lion products and queried against the Lion Localizer database. Using the query sequence, the Lion Localizer generates a listing of exact or partial matches, which are displayed on an interactive map of Africa. This allows for the rapid identification of potential regions and localities where lions have been or are presently being targeted by poachers. By examining the potential provenance of lion samples, the Lion Localizer serves as a valuable resource in the fight against lion poaching. The software is available at https://lionlocalizer.org.

The genomics of ecological flexibility, large brains, and long lives in capuchin monkeys revealed with fecalFACS.

Ecological flexibility, extended lifespans, and large brains have long intrigued evolutionary biologists, and comparative genomics offers an efficient and effective tool for generating new insights into the evolution of such traits. Studies of capuchin monkeys are particularly well situated to shed light on the selective pressures and genetic underpinnings of local adaptation to diverse habitats, longevity, and brain development. Distributed widely across Central and South America, they are inventive and extractive foragers, known for their sensorimotor intelligence. Capuchins have among the largest relative brain size of any monkey and a lifespan that exceeds 50 y, despite their small (3 to 5 kg) body size. We assemble and annotate a de novo reference genome for Cebus imitator Through high-depth sequencing of DNA derived from blood, various tissues, and feces via fluorescence-activated cell sorting (fecalFACS) to isolate monkey epithelial cells, we compared genomes of capuchin populations from tropical dry forests and lowland rainforests and identified population divergence in genes involved in water balance, kidney function, and metabolism. Through a comparative genomics approach spanning a wide diversity of mammals, we identified genes under positive selection associated with longevity and brain development. Additionally, we provide a technological advancement in the use of noninvasive genomics for studies of free-ranging mammals. Our intra- and interspecific comparative study of capuchin genomics provides insights into processes underlying local adaptation to diverse and physiologically challenging environments, as well as the molecular basis of brain evolution and longevity.

A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants.

BACKGROUND: The red junglefowl, the wild outgroup of domestic chickens, has historically served as a reference for genomic studies of domestic chickens. These studies have provided insight into the etiology of traits of commercial importance. However, the use of a single reference genome does not capture diversity present among modern breeds, many of which have accumulated molecular changes due to drift and selection. While reference-based resequencing is well-suited to cataloging simple variants such as single-nucleotide changes and short insertions and deletions, it is mostly inadequate to discover more complex structural variation in the genome. METHODS: We present a pangenome for the domestic chicken consisting of thirty assemblies of chickens from different breeds and research lines. RESULTS: We demonstrate how this pangenome can be used to catalog structural variants present in modern breeds and untangle complex nested variation. We show that alignment of short reads from 100 diverse wild and domestic chickens to this pangenome reduces reference bias by 38%, which affects downstream genotyping results. This approach also allows for the accurate genotyping of a large and complex pair of structural variants at the K feathering locus using short reads, which would not be possible using a linear reference. CONCLUSIONS: We expect that this new paradigm of genomic reference will allow better pinpointing of exact mutations responsible for specific phenotypes, which will in turn be necessary for breeding chickens that meet new sustainability criteria and are resilient to quickly evolving pathogen threats.

Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.