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Phylogenomic data are revolutionizing the field of insect phylogenetics. One of the most tenable and cost-effective methods of generating phylogenomic data is target enrichment, which has resulted in novel phylogenetic hypotheses and revealed new insights into insect evolution. Orthoptera is the most diverse insect order within polyneoptera and includes many evolutionarily and ecologically interesting species. Still, the order as a whole has lagged behind other major insect orders in terms of transitioning to phylogenomics. In this study, we developed an Orthoptera-specific target enrichment (OR-TE) probe set from 80 transcriptomes across Orthoptera. The probe set targets 1828 loci from genes exhibiting a wide range of evolutionary rates. The utility of this new probe set was validated by generating phylogenomic data from 36 orthopteran species that had not previously been subjected to phylogenomic studies. The OR-TE probe set captured an average of 1037 loci across the tested taxa, resolving relationships across broad phylogenetic scales. Our detailed documentation of the probe design and bioinformatics process is intended to facilitate the widespread adoption of this tool., (© 2024. The Author(s).)

As genome resources for wheat (Triticum L.) expand at a rapid pace, it is important to update targeted sequencing tools to incorporate improved sequence assemblies and regions of previously unknown significance. Here, we developed an updated regulatory region enrichment capture for wheat and other Triticeae species. The core target space includes sequences from 2-Kbp upstream of each gene predicted in the Chinese Spring wheat genome (IWGSC RefSeq Annotation v1.0) and regions of open chromatin identified with an assay for transposase-accessible chromatin using sequencing from wheat leaf and root samples. To improve specificity, we aggressively filtered candidate repetitive sequences using a combination of nucleotide basic local alignment search tool (BLASTN) searches to the Triticeae Repetitive Sequence Database (TREP), identification of regions with read over-coverage from previous target enrichment experiments, and k-mer frequency analyses. The final design comprises 216.5 Mbp of predicted hybridization space in hexaploid wheat and showed increased specificity and coverage of targeted sequences relative to previous protocols. Test captures on hexaploid and tetraploid wheat and other diploid cereals show that the assay has broad potential utility for cost-effective promoter and open chromatin resequencing and general-purpose genotyping of various Triticeae species., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Age profiling of archaeological bone assemblages can inform on past animal management practices, but is limited by the fragmentary nature of the fossil record and the lack of universal skeletal markers for age. DNA methylation clocks offer new, albeit challenging, alternatives for estimating the age-at-death of ancient individuals. Here, we take advantage of the availability of a DNA methylation clock based on 31,836 CpG sites and dental age markers in horses to assess age predictions in 84 ancient remains. We evaluate our approach using whole-genome sequencing data and develop a capture assay providing reliable estimates for only a fraction of the cost. We also leverage DNA methylation patterns to assess castration practice in the past. Our work opens for a deeper characterization of past husbandry and ritual practices and holds the potential to reveal age mortality profiles in ancient societies, once extended to human remains., Competing Interests: J.E., A.D., and J.K. are employed by Daicel Arbor Biosciences, who produced the targeted-capture panel and performed the capture laboratory services for this study., (© 2023 The Author(s).)

Cell-free (cf)DNA signatures are quickly becoming the target of choice for non-invasive screening, diagnosis, treatment and monitoring of human tumors. DNA methylation changes occur early in tumorigenesis and are widespread, making cfDNA methylation an attractive cancer biomarker. Already a proven technology for targeted genome sequencing, hybridization probe capture is emerging as a method for high-throughput targeted methylation profiling suitable to liquid biopsy samples. However, to date there are no reports describing the performance of this approach in terms of reproducibility, scalability, and accuracy. In the current study we performed hybridization probe capture using the myBaits ® Custom Methyl-seq kit on 172 plasma samples and standards to evaluate its performance on cfDNA methylation analysis. The myBaits ® assay showed high target recovery (>90%), demonstrated excellent reproducibility between captures ( R 2  = 0.92 on average), and was unaffected by increasing the number of targets in a capture. Finally, myBaits ® accurately replicated 'gold standard' beta values from WGBS (average R 2  = 0.79). The results of this study show that custom targeted methylation sequencing with myBaits ® offers a cost-effective, reliable platform to profile DNA methylation at a set of discrete custom regions, with potential applicability to liquid biopsies for cancer monitoring., (© The Author(s) 2022. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Natural killer (NK) cells are innate lymphocytes that efficiently eliminate cancerous and infected cells. NKp46 is an important NK activating receptor shown to participate in recognition and activation of NK cells against pathogens, tumor cells, virally infected cells, and self-cells in autoimmune conditions, including type I and II diabetes. However, some of the NKp46 ligands are unknown and therefore investigating human NKp46 activity and its critical role in NK cell biology is problematic. We developed a unique anti-human NKp46 monocloncal antibody, denoted hNKp46.02 (02). The 02 mAb can induce receptor internalization and degradation. By binding to a unique epitope on a particular domain of NKp46, 02 lead NKp46 to lysosomal degradation. This downregulation therefore enables the investigation of all NKp46 activities. Indeed, using the 02 mAb we determined NK cell targets which are critically dependent on NKp46 activity, including certain tumor cells lines and human pancreatic beta cells. Most importantly, we showed that a toxin-conjugated 02 inhibits the growth of NKp46-positive cells; thus, exemplifying the potential of 02 in becoming an immunotherapeutic drug to treat NKp46-dependent diseases, such as, type I diabetes and NK and T cell related malignancies., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel.

Dogs were present in the Americas before the arrival of European colonists, but the origin and fate of these precontact dogs are largely unknown. We sequenced 71 mitochondrial and 7 nuclear genomes from ancient North American and Siberian dogs from time frames spanning ~9000 years. Our analysis indicates that American dogs were not derived from North American wolves. Instead, American dogs form a monophyletic lineage that likely originated in Siberia and dispersed into the Americas alongside people. After the arrival of Europeans, native American dogs almost completely disappeared, leaving a minimal genetic legacy in modern dog populations. The closest detectable extant lineage to precontact American dogs is the canine transmissible venereal tumor, a contagious cancer clone derived from an individual dog that lived up to 8000 years ago., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mylodon darwinii is the extinct giant ground sloth named after Charles Darwin, who first collected its remains in South America. We have successfully obtained a high-quality mitochondrial genome at 99-fold coverage using an Illumina shotgun sequencing of a 12 880-year-old bone fragment from Mylodon Cave in Chile. Low level of DNA damage showed that this sample was exceptionally well preserved for an ancient subfossil, probably the result of the dry and cold conditions prevailing within the cave. Accordingly, taxonomic assessment of our shotgun metagenomic data showed a very high percentage of endogenous DNA with 22% of the assembled metagenomic contigs assigned to Xenarthra. Additionally, we enriched over 15 kb of sequence data from seven nuclear exons, using target sequence capture designed against a wide xenarthran dataset. Phylogenetic and dating analyses of the mitogenomic dataset including all extant species of xenarthrans and the assembled nuclear supermatrix unambiguously place Mylodon darwinii as the sister-group of modern two-fingered sloths, from which it diverged around 22 million years ago. These congruent results from both the mitochondrial and nuclear data support the diphyly of the two modern sloth lineages, implying the convergent evolution of their unique suspensory behaviour as an adaption to arboreality. Our results offer promising perspectives for whole-genome sequencing of this emblematic extinct taxon., (© 2018 The Authors.)

Modulation of phase separation temperature (T ph ) for liquid-liquid phase separation of an Fc-fusion protein was studied at pH values below, near, and above its pI where the net charge of the protein was positive, neutral, and negative, respectively, in KF, KCl, KSCN, and MgCl 2 solutions. At the pH value near the pI, the monotonic drop in T ph for all the salt solutions suggests that both the anion and cation can disrupt attractive protein-protein interactions, effectively salting-in the protein. At the pH below or above the pI, the counter-ion neutralizes the net charge on the protein. The neutralization reduces repulsive protein-protein interactions while the co-ion effectively strengthens them. Then, salting-in behavior appears after the completion of charge neutralization. Last, the complex ion-specific modulation on T ph could be rationalized through the rankings of SCN - > Cl - > F - and Mg 2+ > K + for their interactions with the protein throughout the pH conditions., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0-2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.

Herpes simplex virus 1 (HSV1) is a ubiquitous human pathogen that utilizes variable mechanisms to evade immune surveillance. The glycosylphosphatidylinositol (GPI) anchoring pathway is a multistep process in which a myriad of different proteins are covalently attached to a GPI moiety to be presented on the cell surface. Among the different GPI-anchored proteins there are many with immunological importance. We present evidence that the HSV1-encoded miR H8 directly targets PIGT, a member of the protein complex that covalently attaches proteins to GPI in the final step of GPI anchoring. This results in a membrane down-modulation of several different immune-related, GPI-anchored proteins, including ligands for natural killer-activating receptors and the prominent viral restriction factor tetherin. Thus, we suggest that by utilizing just one of dozens of miRNAs encoded by HSV1, the virus can counteract the host immune response at several key points., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Thermal stability of the C H 2 domain for an IgG1 monoclonal antibody and its aggregation kinetics were systematically studied at pH 4.8, below its pI of 8.8 in individual solutions of arginine salts with acetate, glutamate (Glu - ), chloride, and sulfate as the anion, in comparison to sodium chloride and sodium sulfate. Thermal unfolding temperature, T m , an indicator of thermal stability, was measured by both differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). The aggregation kinetics was determined by assessing reversibility for the C H 2 domain in the DSC repetitive scans and then cross-examined by the isothermal aggregation study measured by size exclusion chromatography. The effect of Arg + on the thermal stability and aggregation kinetics of the antibody is shown to be strongly anion-dependent: both ArgAceate and ArgGlu improve the stability, while both Arg 2 SO 4 and ArgCl decrease it. Furthermore, the addition of ArgCl and Arg 2 SO 4 accelerates the aggregation kinetic, but to a lesser extent than the respective Na + salt, suggesting that Arg + binds to the antibody more strongly than Na + . However, the binding of Arg + did not lead to more destabilization of the C H 2 domain by the Arg + salts at low concentrations, comparing to the respective Na + salt. This finding indicates that Arg + prefers the protein surface, rather than the exposed backbone upon unfolding. Furthermore, the change in the ranking for affecting the thermal stability and aggregation kinetics as the salt concentration increases implies the presence of other multiple mechanisms, e.g., cluster formation through the homoion pairing between Arg + molecules and their preferential exclusion from the protein surface, and heteroion pairing between Arg + and SO 4 2- .

Cells in our body can induce hundreds of antiviral genes following virus sensing, many of which remain largely uncharacterized. CEACAM1 has been previously shown to be induced by various innate systems; however, the reason for such tight integration to innate sensing systems was not apparent. Here, we show that CEACAM1 is induced following detection of HCMV and influenza viruses by their respective DNA and RNA innate sensors, IFI16 and RIG-I. This induction is mediated by IRF3, which bound to an ISRE element present in the human, but not mouse, CEACAM1 promoter. Furthermore, we demonstrate that, upon induction, CEACAM1 suppresses both HCMV and influenza viruses in an SHP2-dependent process and achieves this broad antiviral efficacy by suppressing mTOR-mediated protein biosynthesis. Finally, we show that CEACAM1 also inhibits viral spread in ex vivo human decidua organ culture., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Xenarthra (armadillos, sloths, and anteaters) constitutes one of the four major clades of placental mammals. Despite their phylogenetic distinctiveness in mammals, a reference phylogeny is still lacking for the 31 described species. Here we used Illumina shotgun sequencing to assemble 33 new complete mitochondrial genomes, establishing Xenarthra as the first major placental clade to be fully sequenced at the species level for mitogenomes. The resulting data set allowed the reconstruction of a robust phylogenetic framework and timescale that are consistent with previous studies conducted at the genus level using nuclear genes. Incorporating the full species diversity of extant xenarthrans points to a number of inconsistencies in xenarthran systematics and species definition. We propose to split armadillos into two distinct families Dasypodidae (dasypodines) and Chlamyphoridae (euphractines, chlamyphorines, and tolypeutines) to better reflect their ancient divergence, estimated around 42 Ma. Species delimitation within long-nosed armadillos (genus Dasypus) appeared more complex than anticipated, with the discovery of a divergent lineage in French Guiana. Diversification analyses showed Xenarthra to be an ancient clade with a constant diversification rate through time with a species turnover driven by high but constant extinction. We also detected a significant negative correlation between speciation rate and past temperature fluctuations with an increase in speciation rate corresponding to the general cooling observed during the last 15 My. Biogeographic reconstructions identified the tropical rainforest biome of Amazonia and the Guiana Shield as the cradle of xenarthran evolutionary history with subsequent dispersions into more open and dry habitats., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The processes leading up to species extinctions are typically characterized by prolonged declines in population size and geographic distribution, followed by a phase in which populations are very small and may be subject to intrinsic threats, including loss of genetic diversity and inbreeding. However, whether such genetic factors have had an impact on species prior to their extinction is unclear; examining this would require a detailed reconstruction of a species' demographic history as well as changes in genome-wide diversity leading up to its extinction. Here, we present high-quality complete genome sequences from two woolly mammoths (Mammuthus primigenius). The first mammoth was sequenced at 17.1-fold coverage and dates to ∼4,300 years before present, representing one of the last surviving individuals on Wrangel Island. The second mammoth, sequenced at 11.2-fold coverage, was obtained from an ∼44,800-year-old specimen from the Late Pleistocene population in northeastern Siberia. The demographic trajectories inferred from the two genomes are qualitatively similar and reveal a population bottleneck during the Middle or Early Pleistocene, and a more recent severe decline in the ancestors of the Wrangel mammoth at the end of the last glaciation. A comparison of the two genomes shows that the Wrangel mammoth has a 20% reduction in heterozygosity as well as a 28-fold increase in the fraction of the genome that comprises runs of homozygosity. We conclude that the population on Wrangel Island, which was the last surviving woolly mammoth population, was subject to reduced genetic diversity shortly before it became extinct., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Because tissue regeneration deteriorates with age, it is generally assumed that the younger the animal, the better it compensates for tissue damage. We have examined the effect of young age on compensatory proliferation of pancreatic β cells in vivo. Surprisingly, β cells in suckling mice fail to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of β cells for compensatory proliferation is acquired following premature weaning to normal chow, but not to a diet mimicking maternal milk. In addition, weaning coincides with enhanced glucose-stimulated oxidative phosphorylation and insulin secretion from islets. Transcriptome analysis reveals that weaning increases the expression of genes involved in replication licensing, suggesting a mechanism for increased responsiveness to the mitogenic activity of high glucose. We propose that weaning triggers a discrete maturation step of β cells, elevating both the mitogenic and secretory response to glucose., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Natural killer (NK) cells mediate innate immune responses against hazardous cells and are particularly important for the control of human cytomegalovirus (HCMV). NKG2D is a key NK activating receptor that recognizes a family of stress-induced ligands, including MICA, MICB, and ULBP1-6. Notably, most of these ligands are targeted by HCMV proteins and a miRNA to prevent the killing of infected cells by NK cells. A particular highly prevalent MICA allele, MICA ∗ 008, is considered to be an HCMV-resistant "escape variant" that confers advantage to human NK cells in recognizing infected cells. However, here we show that HCMV uses its viral glycoprotein US9 to specifically target MICA ∗ 008 and thus escapes NKG2D attack. The finding that HCMV evolved a protein dedicated to countering a single host allele illustrates the dynamic co-evolution of host and pathogen., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT., (Copyright © 2015 Elsevier Inc. All rights reserved.)

High-throughput sequencing (HTS) has radically altered approaches to human evolutionary research. Recent contributions highlight that HTS is able to reach depths of the human lineage previously thought to be impossible. In this paper, we outline the methodological advances afforded by recent developments in DNA recovery, data output, scalability, speed, and resolution of the current sequencing technology. We review and critically evaluate the 'DNA pipeline' for ancient samples: from DNA extraction, to constructing immortalized sequence libraries, to enrichment strategies (e.g., polymerase chain reaction [PCR] and hybridization capture), and finally, to bioinformatic analyses of sequence data. We argue that continued evaluations and improvements to this process are essential to ensure sequence data validity. Also, we highlight the role of contamination and authentication in ancient DNA-HTS, which is particularly relevant to ancient human genomics, since sequencing the genomes of hominins such as Homo erectus and Homo heidelbergensis may soon be within the realm of possibility., (Copyright © 2014 Elsevier Ltd. All rights reserved.)