Background:Malaria is a mosquito borne disease caused by parasites of the genus Plasmodium. In 2023, the United States (US) experienced nine cases of autochthonous Plasmodium vivax malaria transmission; seven in Florida, one in Texas, and another in Arkansas. These were the first autochthonous cases since 2003 when a cluster was identified in Florida. The aim of this study was to genetically characterize the implicated P. vivax isolates in order to complement epidemiologic investigations of these cases. Methods:A custom Illumina AmpliSeq sequencing panel capturing 495 amplicons was designed. This panel was used to ascertain whether these 2023 cases were related, and assess if they were associated with a single or separate introduction events. Sequence data were hierarchically clustered and a Naïve Bayes classification approach was used to assign genotypes to a probable geographic origin based on 113 'geo-informative' SNPs captured by the panel. Genotypes associated with the 2023 Arkansas, Texas, and Florida cases were clustered alongside those sequenced from archived blood samples from the 2003 Florida case-patients, a set of reference strains, and other travel-associated specimens. Microsatellite analysis was performed on a subset of samples from these autochthonous cases to complement the AmpliSeq analysis. Findings:The 2023 autochthonous Florida cases were genetically linked as were the 2003 Florida cases. The 2023 and 2003 Florida clusters were genetically distinct, and the two Florida clusters were distinct from the 2023 Texas and Arkansas cases, which were also distinct from each other. These genotypes classified to the Central or South American region using the Naïve Bayes classifier, including those from the 2003 cluster. Interpretation:These data support that at least three distinct P. vivax introduction events in the US in 2023, involving parasites possessing genetic signatures consistent with Central or South America. Funding:This work was supported by the National Center for Emerging and Zoonotic Infectious Diseases at the US Centers for Disease Control and Prevention.

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.

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.

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.

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.

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.

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.

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.

We wish to report the synthesis, crystal structures, spectroscopic and electrochemical properties of several new Pt(II) heteroleptic complexes containing the thiacrown, 9S3 (1,4,7-trithiacyclononane) with a series of substituted phenanthroline ligands and related diimine systems. These five ligands are 5,6-dimethyl-1,10-phenanthroline(5,6-Me2-phen), 4,7-dimethyl-1,10-phenanthroline(4,7-Me2-phen), 4,7-diphenyl-1,10-phenanthroline(4,7-Ph2-phen), 2,2′-bipyrimidine(bpm), and pyrazino[2,3-f]quinoxaline or 1,4,5,8-tetraazaphenanthrene(tap). All complexes have the general formula [Pt(9S3)(N2)](PF6)2 (N2=diimine ligand) and form similar structures in which the Pt(II) center is surrounded by a cis arrangement of the two N donors from the diimine chelate and two sulfur atoms from the 9S3 ligand. The third 9S3 sulfur in each structure forms a longer interaction with the platinum resulting in an elongated square pyramidal structure, and this distance is sensitive to the identity of the diimine ligand. In addition, we report the synthesis, structural, electrochemical, and spectroscopic properties of related Pd(II) 9S3 complex with tap. The 195Pt NMR chemical shifts for the six Pt(II) complexes show a value near −3290ppm, consistent with a cis-PtS2N2 coordination sphere although more electron-withdrawing ligands such as tap show resonances shifted by almost 100ppm downfield. The physicochemical properties of the complexes generally follow the electron-donating or withdrawing properties of the phenanthroline substituents.