The first complete genome of the simian malaria parasite Plasmodium brasilianum.

Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like, P. coatneyi, and P. brasilianum. Accurate detection of each species is complicated by their morphological similarities with other Plasmodium species. PCR-based assays offer a solution but require prior knowledge of adequate genomic targets that can distinguish the species. While whole genomes have been published for P. knowlesi, P. cynomolgi, P. simium, and P. inui, no complete genome for P. brasilianum has been available. Previously, we reported a draft genome for P. brasilianum, and here we report the completed genome for P. brasilianum. The genome is 31.4 Mb in size and comprises 14 chromosomes, the mitochondrial genome, the apicoplast genome, and 29 unplaced contigs. The chromosomes consist of 98.4% nucleotide sites that are identical to the P. malariae genome, the closest evolutionarily related species hypothesized to be the same species as P. brasilianum, with 41,125 non-synonymous SNPs (0.0722% of genome) identified between the two genomes. Furthermore, P. brasilianum had 4864 (82.1%) genes that share 80% or higher sequence similarity with 4970 (75.5%) P. malariae genes. This was demonstrated by the nearly identical genomic organization and multiple sequence alignments for the merozoite surface proteins msp3 and msp7. We observed a distinction in the repeat lengths of the circumsporozoite protein (CSP) gene sequences between P. brasilianum and P. malariae. Our results demonstrate a 97.3% pairwise identity between the P. brasilianum and the P. malariae genomes. These findings highlight the phylogenetic proximity of these two species, suggesting that P. malariae and P. brasilianum are strains of the same species, but this could not be fully evaluated with only a single genomic sequence for each species.

Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

CNS Complications in Adult Patients Treated With Extracorporeal Membrane Oxygenation.

OBJECTIVES: To describe the incidence and outcomes of radiologically confirmed acute CNS complications in extracorporeal membrane oxygenation patients at an Australian extracorporeal membrane oxygenation referral center and identify associated patient characteristics. DESIGN: Retrospective cohort study. SETTING: Single-center tertiary institution. PATIENTS: Four-hundred twelve consecutive adult patients supported with extracorporeal membrane oxygenation from 2009 to 2017. RESULTS: Fifty-five patients (13.3%) had a CNS complication confirmed by CT or MRI, including ischemic stroke (7.0%), intracerebral hemorrhage (3.4%), hypoxic ischemic encephalopathy (3.6%), and spinal cord injury (1.2%). CNS complication rates in the venoarterial, venovenous, and veno-pulmonary artery extracorporeal membrane oxygenation subgroups were 18.0%, 4.6%, and 13.6%, respectively. Neurologic complications were independently associated with the use of venoarterial extracorporeal membrane oxygenation (p = 0.002) and renal replacement therapy (p = 0.04). Sixty-five percent of patients with a neurologic complication died during their hospital admission compared with 32% of patients without this complication (p < 0.001). Venoarterial extracorporeal membrane oxygenation, renal replacement therapy, and days of extracorporeal membrane oxygenation support were also associated with hospital mortality and remained so after adjustment in a multivariable regression model (p = 0.01, p < 0.001, and p = 0.003, respectively). CONCLUSIONS: CNS complications appear to occur more frequently in patients requiring circulatory as opposed to respiratory support on extracorporeal membrane oxygenation and are independently associated with mortality. It remains unclear if these complications are causative of a poor outcome or a marker of severity of the underlying condition. Further research is required to better elucidate modifiable or preventable aspects through better patient selection and change in ongoing care.

Evolutionary flexibility in flooding response circuitry in angiosperms.

Flooding due to extreme weather threatens crops and ecosystems. To understand variation in gene regulatory networks activated by submergence, we conducted a high-resolution analysis of chromatin accessibility and gene expression at three scales of transcript control in four angiosperms, ranging from a dryland-adapted wild species to a wetland crop. The data define a cohort of conserved submergence-activated genes with signatures of overlapping cis regulation by four transcription factor families. Syntenic genes are more highly expressed than nonsyntenic genes, yet both can have the cis motifs and chromatin accessibility associated with submergence up-regulation. Whereas the flexible circuitry spans the eudicot-monocot divide, the frequency of specific cis motifs, extent of chromatin accessibility, and degree of submergence activation are more prevalent in the wetland crop and may have adaptive importance.

Methyl-CpG-binding domain 9 (MBD9) is required for H2A.Z incorporation into chromatin at a subset of H2A.Z-enriched regions in the Arabidopsis genome.

The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition.

Changes in chromatin accessibility between Arabidopsis stem cells and mesophyll cells illuminate cell type-specific transcription factor networks.

Cell differentiation is driven by changes in the activity of transcription factors (TFs) and subsequent alterations in transcription. To study this process, differences in TF binding between cell types can be deduced by probing chromatin accessibility. We used cell type-specific nuclear purification followed by the assay for transposase-accessible chromatin (ATAC-seq) to delineate differences in chromatin accessibility and TF regulatory networks between stem cells of the shoot apical meristem (SAM) and differentiated leaf mesophyll cells in Arabidopsis thaliana. Chromatin accessibility profiles of SAM stem cells and leaf mesophyll cells were very similar at a qualitative level, yet thousands of regions having quantitatively different chromatin accessibility were also identified. Analysis of the genomic regions preferentially accessible in each cell type identified hundreds of overrepresented TF-binding motifs, highlighting sets of TFs that are probably important for each cell type. Within these sets, we found evidence for extensive co-regulation of target genes by multiple TFs that are preferentially expressed in each cell type. Interestingly, the TFs within each of these cell type-enriched sets also showed evidence of extensively co-regulating each other. We further found that preferentially accessible chromatin regions in mesophyll cells tended to also be substantially accessible in the stem cells, whereas the converse was not true. This observation suggests that the generally higher accessibility of regulatory elements in stem cells might contribute to their developmental plasticity. This work demonstrates the utility of cell type-specific chromatin accessibility profiling for the rapid development of testable models of regulatory control differences between cell types.

Identification of Open Chromatin Regions in Plant Genomes Using ATAC-Seq.

Identifying and characterizing highly accessible chromatin regions assists in determining the location of genomic regulatory elements and understanding transcriptional regulation. In this chapter, we describe an approach to map accessible chromatin features in plants using the Assay for Transposase-Accessible Chromatin, combined with high-throughput sequencing (ATAC-seq), which was originally developed for cultured animal cells. This technique utilizes a hyperactive Tn5 transposase to cause DNA cleavage and simultaneous insertion of sequencing adapters into open chromatin regions of the input nuclei. The application of ATAC-seq to plant tissue has been challenging due to the difficulty of isolating nuclei sufficiently free of interfering organellar DNA. Here we present two different approaches to purify plant nuclei for ATAC-seq: the INTACT method (Isolation of Nuclei TAgged in specific Cell Types) to isolate nuclei from individual cell types of the plant, and tissue lysis followed by sucrose sedimentation to isolate sufficiently pure total nuclei. We provide detailed instructions for transposase treatment of nuclei isolated using either approach, as well as subsequent preparation of ATAC-seq libraries. Sequencing-ready ATAC-seq libraries can be prepared from plant tissue in as little as one day. The procedures described here are optimized for Arabidopsis thaliana but can also be applied to other plant species.

Profiling of Accessible Chromatin Regions across Multiple Plant Species and Cell Types Reveals Common Gene Regulatory Principles and New Control Modules.

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. We employed the assay for transposase-accessible chromatin (ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and transcription factor (TF) binding sites across each genome. Despite 10-fold variation in intergenic space among species, the majority of open chromatin regions lie within 3 kb upstream of a transcription start site in all species. We find a common set of four TFs that appear to regulate conserved gene sets in the root tips of all four species, suggesting that TF-gene networks are generally conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity as well as many cell-type-specific differences. Analyzing TF binding sites in differentially accessible regions identified a MYB-driven regulatory module unique to the hair cell, which appears to control both cell fate regulators and abiotic stress responses. Our analyses revealed common regulatory principles among species and shed light on the mechanisms producing cell-type-specific transcriptomes during development.