Across its natural and invaded range, Melaleuca quinquenervia, an invasive tree from Australia and Papua New Guinea, produces oils correspondent to two distinct chemotypes. In Australia and Papua New Guinea, the two chemotypes separate geographically: Chemotype 1 occurs north of 25°S and both chemotypes occur south of 25°S. In south Florida, M. quinquenervia also occurs in two different habitats, dry and sandy soils in the west and wetter organic soils in the east. We investigated the effects of two biological control agents, the leaf feeding melaleuca snout weevil (Oxyops vitiosa), and the sap-sucking psyllid (Boreioglycaspis melaleucae) on both chemotypes in both types of habitats. We designed a common garden experiment to investigate the effects of herbivory and irrigation on M. quinquenervia growth between chemotypes. The E-nerolidol chemotype (chemotype 1) grew significantly faster than the viridiflorol chemotype (chemotype 2), regardless of herbivory exposure. Seed production in plants exposed to herbivory was reduced by more than 99% compared to plants with limited herbivory, regardless of chemotype. The final height of insecticide-treated plants was over double that of unsprayed plants and the final trunk biomass was five times greater. However, in our experiments the effects of insecticide on final plant height, final trunk biomass and final branch biomass were greater for viridiflorol than E-nerolidol, suggesting biological control may have a greater impact on viridiflorol trees. Increased irrigation did not affect herbivore numbers but did significantly increase final tree height and final branch weight, and for height this increase was greater for viridiflorol. While our research suggests biological control agents are effective against both chemotypes, they also show that impacts are affected by both chemotype and environment.

Rhesus macaques (RMs) are a vital model for studying human disease and invaluable to pre-clinical vaccine research, particularly for the study of broadly neutralizing antibody responses. Such studies require robust genetic resources for antibody-encoding genes within the immunoglobulin (IG) loci. The complexity of the IG loci has historically made them challenging to characterize accurately. To address this, we developed novel experimental and computational methodologies to generate the largest collection to date of integrated antibody repertoire and long-read genomic sequencing data in 106 Indian origin RMs. We created a comprehensive resource of IG heavy and light chain variable (V), diversity (D), and joining (J) alleles, as well as leader, intronic, and recombination signal sequences (RSSs), including the curation of 1474 novel alleles, unveiling tremendous diversity, and expanding existing IG allele sets by 60%. This publicly available, continually updated resource (https://vdjbase.org/reference_book/Rhesus_Macaque) provides the foundation for advancing RM immunogenomics, vaccine discovery, and translational research.

Background: Acacia auriculiformis (earleaf acacia), an Australian perennial tree, is an increasingly problematic invasive species in Florida. Objective: Create a host range molecular phylogeny by generating test plant patristic distances to earleaf acacia (using rbcL sequences). Methods: DNA was extracted and sequenced from 98 Fabaceae species and an additional 28 sequences were downloaded from NCBI. Results: Molecular phylogenetics and patristic distance inform which plant species should be tested starting with closely related (usually native) plants, especially those threatened or endangered, then extending to less related plants of commercial value. Conclusions: We show the Mimosoideae clade, while monophyletic, appears within the Caesalpinioideae clade. We also suggest 33 mostly threatened or endangered species for the initial test list from the Mimosoideae and Caesalpinioideae, including nine Papilionoideae commercial species. Finally, as a secondary test list, if additional testing is required, we suggest 30 additional Papilionoideae species be added.

The human immunoglobulin heavy chain constant (IGHC) domain of antibodies (Abs) is responsible for effector functions critical to immunity. This domain is encoded by genes in the IGHC locus, where descriptions of genomic diversity remain incomplete. We utilized long-read sequencing to build an IGHC haplotype/variant catalog from 105 individuals of diverse ancestry. We discovered uncharacterized single-nucleotide variants (SNVs) and large structural variants (SVs; n = 7) representing new genes and alleles enriched for non-synonymous substitutions, highlighting potential functional effects. Of the 221 identified IGHC alleles, 192 were novel. SNV, SV, and gene allele/genotype frequencies revealed population differentiation, including (1) hundreds of SNVs in African and East Asian populations exceeding a fixation index (FST) of 0.3 and (2) an IGHG4 haplotype carrying coding variants uniquely enriched in Asian populations. Our results illuminate missing signatures of IGHC diversity and establish a new foundation for investigating IGHC germline variation in Ab function and disease.

Invasive Melaleuca quinquenervia consists of two different chemotypes, E-viridiflorol and E-nerolidol that differentiate geographically in Florida: E-nerolidol dominates on the east coast and E-viridiflorol is more common on the west coast. Both chemotypes are susceptible to attack from three introduced herbivorous biological control insects from Australia: the leaf-feeding weevil, Oxyops vitiosa, the sap sucking psyllid, Boreioglycaspis melaleucae, and the stem-galling midge, Lophodiplosis trifida. Boreioglycaspis melaleucae were collected from areas in Queensland, Australia supporting only the E-viridiflorol chemotype whereas O. vitiosa and L. trifida were collected more broadly in Australia, in areas supporting both chemotypes. Here, we use reciprocal transplants of E-nerolidol and E-viridiflorol from Florida’s east and west coasts, and censuses of naturally occurring trees on both coasts, to determine if biological control agents (a) prefer one chemotype over another or (b) prefer one coastal site over another. Results showed that there was no chemotype preference of O. vitiosa or L. trifida, but we observed a significant preference for E-viridiflorol by B. melaleucae. There were also differences in herbivore densities between east and west coasts that were likely caused by different environmental conditions. We suggest both consideration of chemotype preference as well as impact of environment when selecting biological control agents for field releases.

Genetic diversity within the human immunoglobulin heavy-chain (IGH) locus influences the expressed antibody repertoire and susceptibility to infectious and autoimmune diseases. However, repetitive sequences and complex structural variation pose significant challenges for large-scale characterization. Here, we introduce a method that combines Oxford Nanopore Technologies ultra-long sequencing and adaptive sampling with a bioinformatic pipeline to produce haplotype-resolved, annotated IGH assemblies. Notably, our strategy overcomes prior limitations in phasing resolution, enabling single-contig haplotype assemblies that span the entire IGH locus. We apply this method to four individuals and validate the accuracy of the IGH assemblies using Pacific Biosciences HiFi reads, demonstrating near-complete sequence congruence, with only some residual indel errors. Moreover, when applied to the reference material HG002, our pipeline reveals no base differences and a limited number of indels compared with the telomere-to-telomere genome benchmark across the IGH region. Importantly, in the four individuals, our approach uncovers 28 novel alleles and previously uncharacterized large structural variants, including a 120 kb duplication spanning IGHE to IGHA1 within the IGH constant region (IGHC) and, within the IGHV region, an expanded seven-copy IGHV3-23 gene haplotype. These findings underscore the power of our method to resolve the full complexity of the IGH locus and uncover previously unrecognized variants that may affect immune function and disease susceptibility. Thus, our method provides a strong basis for future immunological research and translational applications.

Classical biological control of weeds is a strategy for managing invasive plants, reassociating coevolved herbivores with their hosts in weeds’ adventive ranges. A modified open field test was conducted to assess the ecological host range of two biological control agents, Floracarus perrepae Knihinicki & Boczek (Prostigmata: Eriophyidae) and Neomusotima conspurcatalis (Warren) (Lepidoptera: Crambidae), on nontarget ferns. These biological control agents are released to aid in the management of Lygodium microphyllum (Cav.) R. Br. (Lygodiaceae), an invasive fern that has had detrimental effects on ecosystems in south and central Florida. We examined nontarget ferns at four sites in Florida for evidence of biological control agent presence/damage, which would be an indication of an expanded ecological host range compared to the physiological host range determined during quarantine studies. The sites were visited multiple times between July 2021 and March 2023, and ferns were examined for evidence of feeding, larval presence, and oviposition. Neither agents nor indicators of their presence were observed on nontarget species. The mite F. perrepae was observed at the three sites on the eastern coast but failed to establish itself at the location on the western coast. In contrast, N. conspurcatalis was found at all locations, although its presence was not consistent across sites over time. Population monitoring indicated both agents were present in sufficient numbers to enable observation of spillover effects, but none were observed. This ecological host range testing validated the predicted physiological host ranges for the two biological control agents, confirming their high specificity to the target fern as observed during laboratory host range testing.

Isoform sequencing (Iso-Seq) uses long-read technology to produce highly accurate full-length reads of mRNA transcripts. Visualization of individual mRNA molecules can reveal new details of transcript variation within understudied portions of mRNA, such as the 5′ untranslated region (UTR). Differential 5′ UTRs may contain motifs, upstream open reading frames (uORFs), and secondary structures that can serve to regulate translation or further indicate changes in promoter usage, where transcriptional control may impact protein expression levels. To begin to explore isoform variation during T-cell activation, we generated the first Iso-Seq reference transcriptome of activated human CD4 T cells. Within this dataset, we discovered many novel splice- and end-variant transcripts. Remarkably, one in every eight genes expressed in our dataset was found to have a notable proportion of transcripts with 5′ UTR lengthened by over 100 bp compared to the longest corresponding UTR within the Gencode dataset. Among these end-variant transcripts, two novel isoforms were identified for CXCR5, a chemokine receptor associated with T follicular helper cell (Tfh) function and differentiation. When investigated in a model cell system, these lengthened UTR conferred reduced transcript stability and, for one of these isoforms, short uORFs introduced by the added length altered protein expression kinetics. This study highlights instances in which current reference databases are incomplete relative to the information obtained by long-read sequencing of intact mRNA. Iso-Seq is thus a promising approach to better understanding the plasticity of promoter usage, alternative splicing, and UTR sequences that influence RNA stability and translation efficiency.

The southern tip of North America coalesces into one of the world’s largest freshwater wetlands, the Everglades, Florida, USA. Though this region is much like an island, home to high biodiversity and endemism, it is also the site of a century of development and associated landscape-scale species invasions. Melaleuca quinquenervia (hereafter melaleuca), a tree native to tropical Australia, was planted extensively throughout south Florida as street trees, levee stabilizers, and later to reduce standing water in marshy areas. Through extensive cooperation with the United States Department of Agriculture’s Australian Biological Control Laboratory, several biological control agents were released, three of which later successfully established. Herein we examine evidence that plant community shifts determined from vegetation surveys and remotely sensed images reflect changes from melaleuca-dominated wetlands (before management) to native communities (after management). Melaleuca-dominated community types decreased from 1990 to 2020 by more than 99%. Vegetation surveys also reflect an increase in cypress wetlands, pinelands, and open water wetlands, all of which were dominated by melaleuca in previous decades. We also found the normalized difference vegetation index (NDVI) increased in melaleuca-invaded wetlands during peak infestations but decreased again after communities recovered to sawgrass-dominated wetlands. These responses are concomitant with the development of integrative management techniques for melaleuca that include mechanical, chemical, and biological control. Our results confirm previous findings that biological control likely augments conventional management methods by limiting recovery of invasive species.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

Disruption of the intestinal microbiome is observed with acute graft-versus-host disease (GVHD) of the lower gastrointestinal (LGI) tract and fecal microbiota transplantation (FMT) has successfully cured steroid-refractory cases. In this open-label, single-arm, pilot study (NCT04139577), third-party, single donor FMT was administered in combination with systemic corticosteroids to participants with high-risk acute LGI GVHD, with a focus on treatment-naïve cases. Participants were scheduled to receive one induction dose (15 capsules/day for 2 consecutive days), followed by 3 weekly maintenance doses, consisting of 15 capsules/dose. The primary endpoint of the study was feasibility, which would be achieved if ≥80% of participants able to swallow ≥40 of the 75 scheduled capsules. Ten participants (9 treatment-naïve; 1 steroid-refractory) were enrolled and treated. The study met the primary endpoint, with 9 of 10 participants completing all eligible doses. Organ-specific LGI complete response rate at Day 28 was 70%. Initial clinical response was observed within 1 week for all responders and clinical responses were durable, without recurrent LGI GVHD in complete responders. Exploratory analyses suggest that alpha diversity increased following FMT. While recipient microbiome composition never achieved a high degree of donor similarity, expansion of donor-derived species and increases in tryptophan metabolites and short-chain fatty acids were observed within the first 7 days after FMT. Investigation into the use of microbiome-targeted interventions earlier in the treatment paradigm for acute LGI GVHD is warranted.

BACKGROUND:Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in a rodent model of alcohol-associated liver disease (ALD). OBJECTIVE:To better understand how PCB126 promoted ALD in our previous model, the current study adopts multiple omics approaches to elucidate potential mechanistic hypotheses. METHODS:Briefly, male C57BL/6J mice were exposed to 0.2mg/kg polychlorinated biphenyl (PCB) 126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were collected and prepared for mRNA sequencing, phosphoproteomics, and inductively coupled plasma mass spectrometry for metals quantification. RESULTS:Principal component analysis showed that PCB126 uniquely modified the transcriptome in EtOH-fed mice. EtOH feeding alone resulted in >4,000 differentially expressed genes (DEGs), and PCB126 exposure resulted in more DEGs in the EtOH-fed group (907 DEGs) in comparison with the pair-fed group (503 DEGs). Top 20 significant gene ontology (GO) biological processes included "peptidyl tyrosine modifications," whereas top 25 significantly decreasing GO molecular functions included "metal/ion/zinc binding." Quantitative, label-free phosphoproteomics and western blot analysis revealed no major significant PCB126 effects on total phosphorylated tyrosine residues in EtOH-fed mice. Quantified hepatic essential metal levels were primarily significantly lower in EtOH-fed mice. PCB126-exposed mice had significantly lower magnesium, cobalt, and zinc levels in EtOH-fed mice. DISCUSSION:Previous work has demonstrated that PCB126 is a modifying factor in metabolic dysfunction-associated steatotic liver disease (MASLD), and our current work suggests that pollutants also modify ALD. PCB126 may, in part, be contributing to the malnutrition aspect of ALD, where metal deficiency is known to contribute and worsen prognosis. https://doi.org/10.1289/EHP14132.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

The high mutation rate of RNA viruses provides viral populations with the ability to adapt to new environments but also makes them vulnerable to extinction due to the deleterious effects of mutations, which is the conceptual basis for the antiviral activity of RNA mutagens. However, there are still gaps in the quantitative understanding of the dynamics between the mutations induced by an RNA mutagen and its effects on viral fitness. To address this, we used Venezuelan Equine Encephalitis Virus (VEEV) and the potent RNA mutagen β-d-N4-hydroxycytidine (NHC) as a model to analyze virus replication competency and mutation frequency following treatment in the total and replication-competent viral populations separately. We found that NHC induced transition mutations in a concentration dependent manner in the total population, while the replication-competent population maintained itself within an increased, yet narrow, mutation spectrum. The incorporation of NHC mainly happened during the positive sense RNA synthesis of VEEV. A growth kinetic analysis of VEEV population treated with NHC pointed to a lower but more diverse distribution in mutational fitness, demonstrating that NHC-induced mutations negatively and broadly affect the fitness of the virus. Together, our study provides mechanistic insight into how RNA mutagens affect viral population landscape and the potential of RNA mutagens as an antiviral strategy for alphaviruses.

Conventional blood-based biomarkers and radiographic imaging are excellent for use in monitoring different aspects of malignant disease, but given their specific shortcomings, their integration with other, complementary markers such as plasma circulating tumor DNA (ctDNA) will be beneficial toward a precision medicine-driven future. Plasma ctDNA analysis utilizes the measurement of cancer-specific molecular alterations in a variety of bodily fluids released by dying tumor cells to monitor and profile response to therapy, and is being employed in several clinical scenarios. Plasma concentrations of ctDNA have been reported to correlate with tumor burden. However, the strength of this association is generally poor and highly variable, confounding the interpretation of longitudinal plasma ctDNA measurements in conjunction with routine radiographic assessments. Herein is discussed what is currently understood with respect to the fundamental characteristics of tumor growth that dictate plasma ctDNA concentrations, with a perspective on its interpretation in conjunction with radiographically determined tumor burden assessments.

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of the IG loci has hindered use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we use long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n = 36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and novel structural variants harboring functional IGKV genes. Among 47 functional IGKV genes, we identify 145 alleles, 67 of which were not previously curated. We report inter-population differences in allele frequencies for 10 IGKV genes, including alleles unique to specific populations within this dataset. We identify haplotypes carrying signatures of gene conversion that associate with SNV enrichment in the IGK distal region, and a haplotype with an inversion spanning the proximal and distal regions. These data provide a critical resource of curated genomic reference information from diverse ancestries, laying a foundation for advancing our understanding of population-level genetic variation in the IGK locus.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

BACKGROUND:Alcohol-associated hepatitis (AH) is the clinical manifestation of alcohol-associated liver disease (ALD). AH is a complex disease encompassing the dysregulation of many cells and cell subpopulations. This study used a hepatic spatial transcriptomic and proteomic approach (10X Genomics Visium) to identify hepatic cell populations and their associated transcriptomic and proteomic alterations in human AH. METHODS:Formalin-fixed paraffin-embedded liver tissue from AH patients (n = 2) and non-ALD controls (donors) (n = 2) were used for Visium spatial transcriptomic and proteomic analysis. RESULTS:AH cell clusters and cell markers were drastically different in regard to tissue pattern and number of cell types compared to non-ALD controls. Cholangiocytes, endothelial cells, macrophages, and stellate cells were more profuse in AH relative to non-ALD controls. Transcriptionally, proliferating cell nuclear antigen-positive (PCNA+) hepatocytes in AH more closely resembled cholangiocytes suggesting they were non-functional hepatocytes derived from cholangiocytes. Furthermore, mitochondria protein-coding genes were reduced in AH versus non-ALD control hepatocytes, suggesting reduced functionality and loss of regenerative mechanisms. Macrophages in AH exhibited elevated gene expression involved in exosomes as compared to non-ALD controls. The most upregulated macrophage genes observed in AH were those involved in exosome trafficking. Gene and protein signatures of disease-associated hepatocytes (ANXA2+/CXCL1+/CEACAM8+) were elevated in AH and could visually identify a pre-malignant lesion. CONCLUSIONS:This study identified global cell type alterations in AH and distinct transcriptomic changes between AH and non-ALD controls. These findings characterize cellular plasticity and profuse transcriptomic and proteomic changes that are apparent in AH and contribute to the identification of novel therapeutics.