Duplication is appreciated as a key source of genomic novelty. In this study, we examined putative enhancers in the human genome to investigate how duplication influences the evolution of these enhancers. We strictly identified a set of 3,336 confident duplicate enhancer pairs and examined their genomic and evolutionary features. We found that, compared to non-duplicated enhancers, duplicated enhancers tend to be longer, more pleiotropic, closer to genes, and harbor greater numbers and more diverse groups of transcription factor binding motifs. These attributes were more pronounced for evolutionarily older duplicate enhancers. Therefore, the regulatory potentials of enhancers may facilitate evolutionary maintenance of duplicated enhancers. Utilizing chimpanzee and rhesus macaque as outgroups, we found that between 30-40% of the examined duplicate enhancers exhibit evidence of asymmetric sequence evolution. Notably, the majority of the “accelerating enhancers” in such pairs gained enhancer activities in novel tissues, particularly in immune-related tissues. Moreover, the accelerating enhancers tended to harbor transcription factor binding motifs previously implicated in human evolution, and enriched in associations with immune functions and stress responses. These findings indicate that duplication may contribute to the proliferation of highly pleiotropic enhancers, as well as gaining novel enhancer activities, and contribute to rapid evolution of the immune system. ### Competing Interest Statement The authors have declared no competing interest.

The mechanisms of X chromosome inactivation suggest fundamental epigenetic differences between the female and male X chromosomes. However, DNA methylation studies often exclude the X chromosomes. In addition, many previous studies relied on techniques that examine non-randomly selected subsets of positions such as array-based methods, rather than assessing the whole X chromosome. Consequently, our understanding of X chromosome DNA methylation lags behind that of autosomes. Here we addressed this gap of knowledge by studying X chromosome DNA methylation using 89 whole genome bisulfite sequencing (WGBS) maps from neurons and oligodendrocytes. Using this unbiased and comprehensive data, we show that DNA methylation of the female X chromosomes is globally reduced (hypomethylated) across the entire chromosome compared to the male X chromosomes and autosomes. On the other hand, the majority of X-linked promoters were more highly methylated (hypermethylated) in females compared to males, consistent with the role of DNA methylation in X chromosome inactivation and dosage compensation. Remarkably, hypermethylation of female X promoters was limited to a group of previously lowly methylated promoters. The other group of highly methylated promoters were both hyper- and hypo- methylated in females with no obvious association with gene expression. Therefore, X chromosome inactivation by DNA methylation was exclusive to a subset of promoters with distinctive epigenetic feature. Apart from this group of promoters, differentially methylated regions in the female and male X chromosomes were dominated by female hypomethylation. Our study furthers the understanding of X-chromosome dosage regulation by DNA methylation on the chromosomal level as well as on individual gene level.

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.

Abstract Enhancers are often studied as noncoding regulatory elements that modulate the precise spatiotemporal expression of genes in a highly tissue-specific manner. This paradigm has been challenged by recent evidence of individual enhancers acting in multiple tissues or developmental contexts. However, the frequency of these enhancers with high degrees of “pleiotropy” out of all putative enhancers is not well understood. Consequently, it is unclear how the variation of enhancer pleiotropy corresponds to the variation in expression breadth of target genes. Here, we use multi-tissue chromatin maps from diverse human tissues to investigate the enhancer–gene interaction architecture while accounting for 1) the distribution of enhancer pleiotropy, 2) the variations of regulatory links from enhancers to target genes, and 3) the expression breadth of target genes. We show that most enhancers are tissue-specific and that highly pleiotropy enhancers account for <1% of all putative regulatory sequences in the human genome. Notably, several genomic features are indicative of increasing enhancer pleiotropy, including longer sequence length, greater number of links to genes, increasing abundance and diversity of encoded transcription factor motifs, and stronger evolutionary conservation. Intriguingly, the number of enhancers per gene remains remarkably consistent for all genes (∼14). However, enhancer pleiotropy does not directly translate to the expression breadth of target genes. We further present a series of Gaussian Mixture Models to represent this organization architecture. Consequently, we demonstrate that a modest trend of more pleiotropic enhancers targeting more broadly expressed genes can generate the observed diversity of expression breadths in the human genome.

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is an iconic example of epigenetic regulation. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here, we examine genome-wide signatures of DNA methylation across fives tissues from a male and female koala ( Phascolarctos cinereus ), and present the first whole-genome, multi-tissue marsupial ‘methylome atlas’. Using these novel data, we elucidate divergent versus common features of representative marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in koalas primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx , the primary lnc RNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-specific (and presumably inactive X chromosome-specific) expression. Finally, we use the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5%) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global outbreak of a coronavirus disease (herein referred to as COVID-19). Other viruses in the same phylogenetic group have been responsible for previous regional outbreaks, including SARS and MERS. SARS-CoV-2 has a zoonotic origin, similar to the causative viruses of these previous outbreaks. The repetitive introduction of animal viruses into human populations resulting in disease outbreaks suggests that similar future epidemics are inevitable. Therefore, understanding the molecular origin and ongoing evolution of SARS-CoV-2 will provide critical insights for preparing for and preventing future outbreaks. A key feature of SARS-CoV-2 is its propensity for genetic recombination across host species boundaries. Consequently, the genome of SARS-CoV-2 harbors signatures of multiple recombination events, likely encompassing multiple species and broad geographic regions. Other regions of the SARS-CoV-2 genome show the impact of purifying selection. The spike (S) protein of SARS-CoV-2, which enables the virus to enter host cells, exhibits signatures of both purifying selection and ancestral recombination events, leading to an effective S protein capable of infecting human and many other mammalian cells. The global spread and explosive growth of the SARS-CoV-2 population (within human hosts) has contributed additional mutational variability into this genome, increasing opportunities for future recombination.

DNA methylation is a critical regulatory mechanism implicated in development, learning, memory, and disease in the human brain. Here we have elucidated DNA methylation changes during recent human brain evolution. We demonstrate dynamic evolutionary trajectories of DNA methylation in cell-type and cytosine-context specific manner. Specifically, DNA methylation in non-CG context, namely CH methylation, has increased (hypermethylation) in neuronal gene bodies during human brain evolution, contributing to human-specific down-regulation of genes and co-expression modules. The effects of CH hypermethylation is particularly pronounced in early development and neuronal subtypes. In contrast, DNA methylation in CG context shows pronounced reduction (hypomethylation) in human brains, notably in cis-regulatory regions, leading to upregulation of downstream genes. We show that the majority of differential CG methylation between neurons and oligodendrocytes originated before the divergence of hominoids and catarrhine monkeys, and harbors strong signal for genetic risk for schizophrenia. Remarkably, a substantial portion of differential CG methylation between neurons and oligodendrocytes emerged in the human lineage since the divergence from the chimpanzee lineage and carries significant genetic risk for schizophrenia. Therefore, recent epigenetic evolution of human cortex has shaped the cellular regulatory landscape and contributed to the increased vulnerability to neuropsychiatric diseases.

Cell-type specific epigenetic modifications are critical for brain development and neuropsychiatric diseases. Here we elucidate evolutionary origins of neuron- and oligodendrocyte-specific DNA methylation in human prefrontal cortex, and demonstrate dynamic and distinctive changes of CG and CH methylation. We show that the human brain has experienced pronounced reduction of CG methylation during evolution, which significantly contributed to cell-type specific active regulatory regions. On the other hand, a substantial increase of CH methylation occurred during human brain evolution, associated with fine-tuning expression in development and neuronal subtypes. The majority of differential CG methylation between neurons and oligodendrocytes originated before the divergence of hominoids and catarrhine monkeys, and carries strong signal for genetic risk for schizophrenia. Remarkably, a substantial portion of differential CG methylation between neurons and oligodendrocytes emerged in the human lineage and harbors additional genetic risk for schizophrenia, implicating epigenetic evolution of human cortex in increased vulnerability to neuropsychiatric diseases.

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is well characterized in eutherian mammals. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here we examine genome-wide signatures of DNA methylation from methylation maps across fives tissues from a male and female koala ( Phascolarctos cinereus ) and present the first whole genome, multi-tissue marsupial “methylome atlas.” Using these novel data, we elucidate divergent versus common features of marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in marsupials primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx , the primary lnc RNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-(and presumably inactive X chromosome-) specific expression. Finally, we utilize the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5 %) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.### Competing Interest StatementThe authors have declared no competing interest.

As more countries provide free health care, pharmaceutical reimbursement lists are becoming a concern, especially in low- and middle-income countries. In 2007, Nepal decreed that health is a human right and began basic health coverage for a target group of the poor, destitute, elderly, and disabled. The Ministry of Health and Population (MoHP) also provided 40 drugs without cost to all citizens through the Free Drugs List (FDL) program. The FDL was later expanded from 40 to 70 drugs; however, the process of review and update remains unclear. To propose a mechanism for future development of the FDL, we conducted a document review and in-depth consultations with representatives from the MoHP and the World Health Organization Country Office during a workshop in Kathmandu. The FDL suffers from lack of an appropriate process, gaps between the listed drugs and Nepal’s burden of disease, and no consideration of the unit costs or cost-effectiveness of drugs included in the list. We propose a new drug selection process that is a variant of the health technology assessment process. This process can be applied not only in Nepal but also in other resource-limited countries that wish to ensure their citizens’ access to essential medicines through a pharmaceutical reimbursement list.

The rise of multi-drug-resistant (MDR) bacteria has spurred renewed interest in the use of bacteriophages in therapy. However, mechanisms contributing to phage-mediated bacterial clearance in an animal host remain unclear. We investigated the effects of host immunity on the efficacy of phage therapy for acute pneumonia caused by MDR Pseudomonas aeruginosa in a mouse model. Comparing efficacies of phage-curative and prophylactic treatments in healthy immunocompetent, MyD88-deficient, lymphocyte-deficient, and neutrophil-depleted murine hosts revealed that neutrophil-phage synergy is essential for the resolution of pneumonia. Population modeling of in vivo results further showed that neutrophils are required to control both phage-sensitive and emergent phage-resistant variants to clear infection. This "immunophage synergy" contrasts with the paradigm that phage therapy success is largely due to bacterial permissiveness to phage killing. Lastly, therapeutic phages were not cleared by pulmonary immune effector cells and were immunologically well tolerated by lung tissues.

—The economic and health burden of noncommunicable diseases (NCDs) is significant globally. To counteract this problem, the World Health Organization (WHO) introduced the Package of Essential Noncommunicable disease (PEN) interventions. Several countries, including Indonesia, implemented the PEN program. To assess the value of the investment in the current program, an economic evaluation of the program was conducted with collaboration between the Ministry of Health in Indonesia, the WHO, and the International Decision Support Initiative (iDSI). Even in low- and middle-income countries (LMICs) such as Indonesia where there is lack of data and health technology assessment (HTA) expertise, the study aims not only to inform policy but to build HTA capacity in the country through the working partnership between international HTA experts and local partners.This study evaluated the delivery of screening and treatment for diabetes and hypertension, which are part of NCD interventions in the PEN program. Several screening strategies were compared to explore the options for improving the current PEN program. The findings show that implementing the PEN program is better than a base case of no policy in place, though it can be improved through a targeted screening policy of high-risk groups of population aged 40 and above (as opposed to screening for 15 years old and above as is the current practice). Adopting the recommended policy is a major challenge to policy makers due to a potential negative public perception of the disinvestment from an option that yields higher health outcomes. However, this study demonstrates that with the same budget currently invested in the program, the changes proposed will result in improvements on the current low uptake and poor coverage, thus yielding cost savings for the government and a possibility to reallocate resources to the country's priority health concerns, consequently leading to better health outcomes.