Risk factors for disease progression and treatment goals in polycythemia vera.

Polycythemia vera is a Philadelphia chromosome-negative myeloproliferative neoplasm characterized by the clonal proliferation of hematopoietic cells, leading to the overproduction of erythrocytes and the elaboration of inflammatory cytokines. Management is aimed at reducing the risk of thromboembolic events, alleviating the symptom burden, decreasing splenomegaly, and potentially mitigating the risk of disease progression. Existing treatment options include therapeutic phlebotomy and cytoreductive agents including hydroxyurea, pegylated recombinant interferon alpha 2a, ropegylated recombinant interferon alpha 2b, and ruxolitinib. We review risk factors for both thrombotic events and disease progression in patients with polycythemia vera. We discuss existing and novel therapeutic approaches to mitigate the risk of disease-related complications and progression.

Lone star ticks (Acari: Ixodidae) infected with Bourbon virus in New Jersey, USA.

Lone star ticks (Amblyomma americanum L.) are expanding within the northeast United States, a region historically focused on Ixodes scapularis-transmitted diseases. In Monmouth County, NJ, the shift has been dramatic, and lone star ticks now vastly outnumber blacklegged ticks. As a result, there is an enhanced need to focus on the potential health risks of A. americanum-transmitted pathogens, such as the emerging Heartland (HRTV) and Bourbon (BRBV) viruses. We screened 1,205 nymphal lone star ticks for HRTV and BRBV using RT-qPCR assays and detected BRBV in 3 ticks collected in Monmouth County, NJ, in 2021. Additionally, we sequenced a complete BRBV genome from a single infected specimen, finding 99.4% identity with human pathogenic isolates from the eastern-central United States. Our results have important public health implications for a region only recently becoming aware of public health risks posed by lone star ticks. Of note, we report successful detection of viral RNA in samples that were stored and intended for DNA preservation, for example, kept in ethanol at room temperature, which may reduce barriers for public health agencies seeking to expand their tick testing to include viruses.

Transcriptional response of individual Hawaiian Culex quinquefasciatus mosquitoes to the avian malaria parasite Plasmodium relictum.

BACKGROUND: Plasmodium parasites that cause bird malaria occur in all continents except Antarctica and are primarily transmitted by mosquitoes in the genus Culex. Culex quinquefasciatus, the mosquito vector of avian malaria in Hawai'i, became established in the islands in the 1820s. While the deadly effects of malaria on endemic bird species have been documented for many decades, vector-parasite interactions in avian malaria systems are relatively understudied. METHODS: To evaluate the gene expression response of mosquitoes exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i, offspring of wild-collected Hawaiian Cx. quinquefasciatus were fed on a domestic canary infected with a fresh isolate of Plasmodium relictum GRW4 from a wild-caught Hawaiian honeycreeper. Control mosquitoes were fed on an uninfected canary. Transcriptomes of five infected and three uninfected individual mosquitoes were sequenced at each of three stages of the parasite life cycle: 24 h post feeding (hpf) during ookinete invasion; 5 days post feeding (dpf) when oocysts are developing; 10 dpf when sporozoites are released and invade the salivary glands. RESULTS: Differential gene expression analyses showed that during ookinete invasion (24 hpf), genes related to oxidoreductase activity and galactose catabolism had lower expression levels in infected mosquitoes compared to controls. Oocyst development (5 dpf) was associated with reduced expression of a gene with a predicted innate immune function. At 10 dpf, infected mosquitoes had reduced expression levels of a serine protease inhibitor, and further studies should assess its role as a Plasmodium agonist in C. quinquefasciatus. Overall, the differential gene expression response of Hawaiian Culex exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i was low, but more pronounced during ookinete invasion. CONCLUSIONS: This is the first analysis of the transcriptional responses of vectors to malaria parasites in non-mammalian systems. Interestingly, few similarities were found between the response of Culex infected with a bird Plasmodium and those reported in Anopheles infected with human Plasmodium. The relatively small transcriptional changes observed in mosquito genes related to immune response and nutrient metabolism support conclusions of low fitness costs often documented in experimental challenges of Culex with avian Plasmodium.

A gene-based capture assay for surveying patterns of genetic diversity and insecticide resistance in a worldwide group of invasive mosquitoes.

Understanding patterns of diversification, genetic exchange, and pesticide resistance in arthropod disease vectors is necessary for effective population management. With the availability of next-generation sequencing technologies, one of the best approaches for surveying such patterns involves the simultaneous genotyping of many samples for a large number of genetic markers. To this end, the targeting of gene sequences of known function can be a cost-effective strategy. One insect group of substantial health concern are the mosquito taxa that make up the Culex pipiens complex. Members of this complex transmit damaging arboviruses and filariae worms to humans, as well as other pathogens such as avian malaria parasites that are detrimental to birds. Here we describe the development of a targeted, gene-based assay for surveying genetic diversity and population structure in this mosquito complex. To test the utility of this assay, we sequenced samples from several members of the complex, as well as from distinct populations of the relatively under-studied Culex quinquefasciatus. The data generated was then used to examine taxonomic divergence and population clustering between and within these mosquitoes. We also used this data to investigate genetic variants present in our samples that had previously been shown to correlate with insecticide-resistance. Broadly, our gene capture approach successfully enriched the genomic regions of interest, and proved effective for facilitating examinations of taxonomic divergence and geographic clustering within the Cx. pipiens complex. It also allowed us to successfully survey genetic variation associated with insecticide resistance in Culex mosquitoes. This enrichment protocol will be useful for future studies that aim to understand the genetic mechanisms underlying the evolution of these ubiquitous and increasingly damaging disease vectors.

Comparative hologenomics of two Ixodes scapularis tick populations in New Jersey.

Tick-borne diseases, such as those transmitted by the blacklegged tick Ixodes scapularis, are a significant and growing public health problem in the US. There is mounting evidence that co-occurring non-pathogenic microbes can also impact tick-borne disease transmission. Shotgun metagenome sequencing enables sampling of the complete tick hologenome-the collective genomes of the tick and all of the microbial species contained therein, whether pathogenic, commensal or symbiotic. This approach simultaneously uncovers taxonomic composition and allows the detection of intraspecific genetic variation, making it a useful tool to compare spatial differences across tick populations. We evaluated this approach by comparing hologenome data from two tick samples (N = 6 ticks per location) collected at a relatively fine spatial scale, approximately 23 km apart, within a single US county. Several intriguing variants in the data between the two sites were detected, including polymorphisms in both in the tick's own mitochondrial DNA and that of a rickettsial endosymbiont. The two samples were broadly similar in terms of the microbial species present, including multiple known tick-borne pathogens (Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum), filarial nematodes, and Wolbachia and Babesia species. We assembled the complete genome of the rickettsial endosymbiont (most likely Rickettsia buchneri) from both populations. Our results provide further evidence for the use of shotgun metagenome sequencing as a tool to compare tick hologenomes and differentiate tick populations across localized spatial scales.

Analysis of an improved Cyanophora paradoxa genome assembly.

Glaucophyta are members of the Archaeplastida, the founding group of photosynthetic eukaryotes that also includes red algae (Rhodophyta), green algae, and plants (Viridiplantae). Here we present a high-quality assembly, built using long-read sequences, of the ca. 100 Mb nuclear genome of the model glaucophyte Cyanophora paradoxa. We also conducted a quick-freeze deep-etch electron microscopy (QFDEEM) analysis of C. paradoxa cells to investigate glaucophyte morphology in comparison to other organisms. Using the genome data, we generated a resolved 115-taxon eukaryotic tree of life that includes a well-supported, monophyletic Archaeplastida. Analysis of muroplast peptidoglycan (PG) ultrastructure using QFDEEM shows that PG is most dense at the cleavage-furrow. Analysis of the chlamydial contribution to glaucophytes and other Archaeplastida shows that these foreign sequences likely played a key role in anaerobic glycolysis in primordial algae to alleviate ATP starvation under night-time hypoxia. The robust genome assembly of C. paradoxa significantly advances knowledge about this model species and provides a reference for exploring the panoply of traits associated with the anciently diverged glaucophyte lineage.

Genome analysis of the rice coral Montipora capitata.

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai'i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M. capitata. These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.

Discovery of SCORs: Anciently derived, highly conserved gene-associated repeats in stony corals.

Stony coral (Scleractinia) genomes are still poorly explored and many questions remain about their evolution and contribution to the success and longevity of reefs. We analyzed transcriptome and genome data from Montipora capitata, Acropora digitifera, and transcriptome data from 20 other coral species. To our surprise, we found highly conserved, anciently derived, Scleractinia COral-specific Repeat families (SCORs) that are abundant in all the studied lineages. SCORs form complex secondary structures and are located in untranslated regions and introns, but most abundant in intergenic DNA. These repeat families have undergone frequent duplication and degradation, suggesting a 'boom and bust' cycle of invasion and loss. We speculate that due to their surprisingly high sequence identities across deeply diverged corals, physical association with genes, and dynamic evolution, SCORs might have adaptive functions in corals that need to be explored using population genomic and function-based approaches.

Divergent evolutionary histories of DNA markers in a Hawaiian population of the coral Montipora capitata.

We investigated intra- and inter-colony sequence variation in a population of the dominant Hawaiian coral Montipora capitata by analyzing marker gene and genomic data. Ribosomal ITS1 regions showed evidence of a reticulate history among the colonies, suggesting incomplete rDNA repeat homogenization. Analysis of the mitochondrial genome identified a major (M. capitata) and a minor (M. flabellata) haplotype in single polyp-derived sperm bundle DNA with some colonies containing 2-3 different mtDNA haplotypes. In contrast, Pax-C and newly identified single-copy nuclear genes showed either no sequence differences or minor variations in SNP frequencies segregating among the colonies. Our data suggest past mitochondrial introgression in M. capitata, whereas nuclear single-copy loci show limited variation, highlighting the divergent evolutionary histories of these coral DNA markers.

Analysis of Gambierdiscus transcriptome data supports ancient origins of mixotrophic pathways in dinoflagellates.

Toxic dinoflagellates pose serious threats to human health and to fisheries. The genus Gambierdiscus is significant in this respect because its members produce ciguatoxin that accumulates in predominantly tropical marine food webs and leads to ciguatera fish poisoning. Understanding the biology of toxic dinoflagellates is crucial to developing control strategies. To this end, we generated a de novo transcriptome library from G. caribaeus and studied its growth under different culture conditions to elucidate pathways of carbon (C) and nitrogen (N) utilization. We also gathered available dinoflagellate transcriptome data to trace the evolutionary history of C and N pathways in this phylum. We find that rather than being specific adaptations to the epiphytic lifestyle in G. caribaeus, the majority of dinoflagellates share a large array of genes that putatively confer mixotrophy and the ability to use N via the ornithine-urea cycle and nitric oxide synthase production. These results suggest that prior to plastid endosymbiosis, the dinoflagellate ancestor possessed complex pathways that linked metabolism, intercellular signaling, and stress responses to environmental cues that have been maintained by extant photosynthetic species. This metabolic flexibility likely explains the success of dinoflagellates in marine ecosystems and may presage difficulties in controlling the spread of toxic species.

Phylogenomic analysis uncovers the evolutionary history of nutrition and infection mode in rice blast fungus and other Magnaporthales.

The order Magnaporthales (Ascomycota, Fungi) includes devastating pathogens of cereals, such as the rice blast fungus Pyricularia (Magnaporthe) oryzae, which is a model in host-pathogen interaction studies. Magnaporthales also includes saprotrophic species associated with grass roots and submerged wood. Despite its scientific and economic importance, the phylogenetic position of Magnaporthales within Sordariomycetes and the interrelationships of its constituent taxa, remain controversial. In this study, we generated novel transcriptome data from 21 taxa that represent key Magnaporthales lineages of different infection and nutrition modes and phenotypes. Phylogenomic analysis of >200 conserved genes allowed the reconstruction of a robust Sordariomycetes tree of life that placed the monophyletic group of Magnaporthales sister to Ophiostomatales. Among Magnaporthales, three major clades were recognized: 1) an early diverging clade A comprised of saprotrophs associated with submerged woods; 2) clade B that includes the rice blast fungus and other pathogens that cause blast diseases of monocot plants. These species infect the above-ground tissues of host plants using the penetration structure, appressorium; and 3) clade C comprised primarily of root-associated species that penetrate the root tissue with hyphopodia. The well-supported phylogenies provide a robust framework for elucidating evolution of pathogenesis, nutrition modes, and phenotypic characters in Magnaporthales.

Antiviral activity of transiently expressed IFN-kappa is cell-associated.

Most type I interferons (IFNs) are expressed by the majority of cell types in response to viral infection. In contrast, IFN-kappa has been reported to have a cellular distribution limited to keratinocytes and certain lymphoid cell populations. Recombinant expressed IFN-kappa has been shown previously to possess weak antiviral activity when directly compared with IFN-beta. In order to expand on the antiviral potential of IFN-kappa, we transiently transfected human cell lines to circumvent the need to purify recombinant proteins and to avoid the possible loss of biologic activity by the purification process. We evaluated the transcriptional signaling and antiviral activity of IFN-kappa in parallel with IFN-alpha2b with mammalian expression vectors to express each protein transiently. Both IFN-kappa and IFN-alpha2b exhibited comparable transcriptional and antiviral activities. However, in contrast to IFN-alpha2b transcriptional signaling and antiviral activity, IFN-kappa activity was not detectable in conditioned cell culture medium. Subsequent experiments revealed there was a direct relationship between IFN-kappa-expressing cells and antiviral activity. These results were confirmed in immunocytochemical studies. Furthermore, IFN-kappa exhibited cell-associated antiviral activity against a hepatitis C virus (HCV) replicon cell line. This novel IFN signaling strategy may represent an important distinct and divergent mechanism for limiting viral infections.

Biological evaluation and interconversion studies of rotamers of SCH 351125, an orally bioavailable CCR5 antagonist.

The separation and biological evaluation of rotamers as well as interconversion studies on rotamers of our clinical candidate SCH 351125 are described.