Improvements in viral gene annotation using large language models and soft alignments.

BACKGROUND: The annotation of protein sequences in public databases has long posed a challenge in molecular biology. This issue is particularly acute for viral proteins, which demonstrate limited homology to known proteins when using alignment, k-mer, or profile-based homology search approaches. A novel methodology employing Large Language Models (LLMs) addresses this methodological challenge by annotating protein sequences based on embeddings. RESULTS: Central to our contribution is the soft alignment algorithm, drawing from traditional protein alignment but leveraging embedding similarity at the amino acid level to bypass the need for conventional scoring matrices. This method not only surpasses pooled embedding-based models in efficiency but also in interpretability, enabling users to easily trace homologous amino acids and delve deeper into the alignments. Far from being a black box, our approach provides transparent, BLAST-like alignment visualizations, combining traditional biological research with AI advancements to elevate protein annotation through embedding-based analysis while ensuring interpretability. Tests using the Virus Orthologous Groups and ViralZone protein databases indicated that the novel soft alignment approach recognized and annotated sequences that both blastp and pooling-based methods, which are commonly used for sequence annotation, failed to detect. CONCLUSION: The embeddings approach shows the great potential of LLMs for enhancing protein sequence annotation, especially in viral genomics. These findings present a promising avenue for more efficient and accurate protein function inference in molecular biology.

Ubiquitous, B12-dependent virioplankton utilizing ribonucleotide-triphosphate reductase demonstrate interseasonal dynamics and associate with a diverse range of bacterial hosts in the pelagic ocean.

Through infection and lysis of their coexisting bacterial hosts, viruses impact the biogeochemical cycles sustaining globally significant pelagic oceanic ecosystems. Currently, little is known of the ecological interactions between lytic viruses and their bacterial hosts underlying these biogeochemical impacts at ecosystem scales. This study focused on populations of lytic viruses carrying the B12-dependent Class II monomeric ribonucleotide reductase (RNR) gene, ribonucleotide-triphosphate reductase (Class II RTPR), documenting seasonal changes in pelagic virioplankton and bacterioplankton using amplicon sequences of Class II RTPR and the 16S rRNA gene, respectively. Amplicon sequence libraries were analyzed using compositional data analysis tools that account for the compositional nature of these data. Both virio- and bacterioplankton communities responded to environmental changes typically seen across seasonal cycles as well as shorter term upwelling-downwelling events. Defining Class II RTPR-carrying viral populations according to major phylogenetic clades proved a more robust means of exploring virioplankton ecology than operational taxonomic units defined by percent sequence homology. Virioplankton Class II RTPR populations showed positive associations with a broad phylogenetic diversity of bacterioplankton including dominant taxa within pelagic oceanic ecosystems such as Prochlorococcus and SAR11. Temporal changes in Class II RTPR virioplankton, occurring as both free viruses and within infected cells, indicated possible viral-host pairs undergoing sustained infection and lysis cycles throughout the seasonal study. Phylogenetic relationships inferred from Class II RTPR sequences mirrored ecological patterns in virio- and bacterioplankton populations demonstrating possible genome to phenome associations for an essential viral replication gene.

Gallionellaceae pangenomic analysis reveals insight into phylogeny, metabolic flexibility, and iron oxidation mechanisms.

IMPORTANCE: Neutrophilic iron-oxidizing bacteria (FeOB) produce copious iron (oxyhydr)oxides that can profoundly influence biogeochemical cycles, notably the fate of carbon and many metals. To fully understand environmental microbial iron oxidation, we need a thorough accounting of iron oxidation mechanisms. In this study, we show the Gallionellaceae FeOB genomes encode both characterized iron oxidases as well as uncharacterized multiheme cytochromes (MHCs). MHCs are predicted to transfer electrons from extracellular substrates and likely confer metabolic capabilities that help Gallionellaceae occupy a range of different iron- and mineral-rich niches. Gallionellaceae appear to specialize in iron oxidation, so it would be advantageous for them to have multiple mechanisms to oxidize various forms of iron, given the many iron minerals on Earth, as well as the physiological and kinetic challenges faced by FeOB. The multiple iron/mineral oxidation mechanisms may help drive the widespread ecological success of Gallionellaceae.

Metagenomic Analysis of a Concrete Bridge Reveals a Microbial Community Dominated by Halophilic Bacteria and Archaea.

Concrete hosts a small but diverse microbiome that changes over time. Shotgun metagenomic sequencing would enable assessment of both the diversity and function of the microbial community in concrete, but a number of unique challenges make this difficult for concrete samples. The high concentration of divalent cations in concrete interferes with nucleic acid extraction, and the extremely low biomass in concrete means that DNA from laboratory contamination may be a large fraction of the sequence data. Here, we develop an improved method for DNA extraction from concrete, with higher yield and lower laboratory contamination. To show that this method provides DNA of sufficient quality and quantity to do shotgun metagenomic sequencing, DNA was extracted from a sample of concrete obtained from a road bridge and sequenced with an Illumina MiSeq system. This microbial community was dominated by halophilic Bacteria and Archaea, with enriched functional pathways related to osmotic stress responses. Although this was a pilot-scale effort, we demonstrate that metagenomic sequencing can be used to characterize microbial communities in concrete and that older concrete structures may host different microbes than recently poured concrete. IMPORTANCE Prior work on the microbial communities of concrete focused on the surfaces of concrete structures such as sewage pipes or bridge pilings, where thick biofilms were easy to observe and sample. Because the biomass inside concrete is so low, more recent analyses of the microbial communities inside concrete used amplicon sequencing methods to describe those communities. However, to understand the activity and physiology of microbes in concrete, or to develop living infrastructure, we must develop more direct methods of community analysis. The method developed here for DNA extraction and metagenomic sequencing can be used for analysis of microbial communities inside concrete and can likely be adapted for other cementitious materials.

Functional Annotation Routines Used by ABRF Bioinformatics Core Facilities - Observations, Comparisons, and Considerations.

The functional annotation of gene lists is a common analysis routine required for most genomics experiments, and bioinformatics core facilities must support these analyses. In contrast to methods such as the quantitation of RNA-Seq reads or differential expression analysis, our research group noted a lack of consensus in our preferred approaches to functional annotation. To investigate this observation, we selected 4 experiments that represent a range of experimental designs encountered by our cores and analyzed those data with 6 tools used by members of the Association of Biomolecular Resource Facilities (ABRF) Genomic Bioinformatics Research Group (GBIRG). To facilitate comparisons between tools, we focused on a single biological result for each experiment. These results were represented by a gene set, and we analyzed these gene sets with each tool considered in our study to map the result to the annotation categories presented by each tool. In most cases, each tool produces data that would facilitate identification of the selected biological result for each experiment. For the exceptions, Fisher's exact test parameters could be adjusted to detect the result. Because Fisher's exact test is used by many functional annotation tools, we investigated input parameters and demonstrate that, while background set size is unlikely to have a significant impact on the results, the numbers of differentially expressed genes in an annotation category and the total number of differentially expressed genes under consideration are both critical parameters that may need to be modified during analyses. In addition, we note that differences in the annotation categories tested by each tool, as well as the composition of those categories, can have a significant impact on results.

Spontaneously Produced Lysogenic Phages Are an Important Component of the Soybean Bradyrhizobium Mobilome.

The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity's nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation (nod) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture.

Gallionellaceae pangenomic analysis reveals insight into phylogeny, metabolic flexibility, and iron oxidation mechanisms.

The iron-oxidizing Gallionellaceae drive a wide variety of biogeochemical cycles through their metabolisms and biominerals. To better understand the environmental impacts of Gallionellaceae, we need to improve our knowledge of their diversity and metabolisms, especially any novel iron oxidation mechanisms. Here, we used a pangenomic analysis of 103 genomes to resolve Gallionellaceae phylogeny and explore the range of genomic potential. Using a concatenated ribosomal protein tree and key gene patterns, we determined Gallionellaceae has four genera, divided into two groups-iron-oxidizing bacteria (FeOB) Gallionella, Sideroxydans, and Ferriphaselus with known iron oxidases (Cyc2, MtoA) and nitrite-oxidizing bacteria (NOB) Candidatus Nitrotoga with nitrite oxidase (Nxr). The FeOB and NOB have similar electron transport chains, including genes for reverse electron transport and carbon fixation. Auxiliary energy metabolisms including S oxidation, denitrification, and organotrophy were scattered throughout the Gallionellaceae FeOB. Within FeOB, we found genes that may represent adaptations for iron oxidation, including a variety of extracellular electron uptake (EEU) mechanisms. FeOB genomes encoded more predicted c-type cytochromes overall, notably more multiheme c-type cytochromes (MHCs) with >10 CXXCH motifs. These include homologs of several predicted outer membrane porin-MHC complexes, including MtoAB and Uet. MHCs are known to efficiently conduct electrons across longer distances and function across a wide range of redox potentials that overlap with mineral redox potentials, which can help expand the range of usable iron substrates. Overall, the results of pangenome analyses suggest that the Gallionellaceae genera Gallionella, Sideroxydans, and Ferriphaselus are primarily iron oxidizers, capable of oxidizing dissolved Fe2+ as well as a range of solid iron or other mineral substrates.

Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS.

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim's Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu2+ and Zn2+. Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu2+ concentrations and variability in Zn2+ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (106-108 cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

Novel Viral DNA Polymerases From Metagenomes Suggest Genomic Sources of Strand-Displacing Biochemical Phenotypes.

Viruses are the most abundant and diverse biological entities on the planet and constitute a significant proportion of Earth's genetic diversity. Most of this diversity is not represented by isolated viral-host systems and has only been observed through sequencing of viral metagenomes (viromes) from environmental samples. Viromes provide snapshots of viral genetic potential, and a wealth of information on viral community ecology. These data also provide opportunities for exploring the biochemistry of novel viral enzymes. The in vitro biochemical characteristics of novel viral DNA polymerases were explored, testing hypothesized differences in polymerase biochemistry according to protein sequence phylogeny. Forty-eight viral DNA Polymerase I (PolA) proteins from estuarine viromes, hot spring metagenomes, and reference viruses, encompassing a broad representation of currently known diversity, were synthesized, expressed, and purified. Novel functionality was shown in multiple PolAs. Intriguingly, some of the estuarine viral polymerases demonstrated moderate to strong innate DNA strand displacement activity at high enzyme concentration. Strand-displacing polymerases have important technological applications where isothermal reactions are desirable. Bioinformatic investigation of genes neighboring these strand displacing polymerases found associations with SNF2 helicase-associated proteins. The specific function of SNF2 family enzymes is unknown for prokaryotes and viruses. In eukaryotes, SNF2 enzymes have chromatin remodeling functions but do not separate nucleic acid strands. This suggests the strand separation function may be fulfilled by the DNA polymerase for viruses carrying SNF2 helicase-associated proteins. Biochemical data elucidated from this study expands understanding of the biology and ecological behavior of unknown viruses. Moreover, given the numerous biotechnological applications of viral DNA polymerases, novel viral polymerases discovered within viromes may be a rich source of biological material for further in vitro DNA amplification advancements.

Towards an integrative view of virus phenotypes.

Understanding how phenotypes emerge from genotypes is a foundational goal in biology. As challenging as this task is when considering cellular life, it is further complicated in the case of viruses. During replication, a virus as a discrete entity (the virion) disappears and manifests itself as a metabolic amalgam between the virus and the host (the virocell). Identifying traits that unambiguously constitute a virus's phenotype is straightforward for the virion, less so for the virocell. Here, we present a framework for categorizing virus phenotypes that encompasses both virion and virocell stages and considers functional and performance traits of viruses in the context of fitness. Such an integrated view of virus phenotype is necessary for comprehensive interpretation of viral genome sequences and will advance our understanding of viral evolution and ecology.

Unraveling Fe(II)-Oxidizing Mechanisms in a Facultative Fe(II) Oxidizer, Sideroxydans lithotrophicus Strain ES-1, via Culturing, Transcriptomics, and Reverse Transcription-Quantitative PCR.

Sideroxydans lithotrophicus ES-1 grows autotrophically either by Fe(II) oxidation or by thiosulfate oxidation, in contrast to most other isolates of neutrophilic Fe(II)-oxidizing bacteria (FeOB). This provides a unique opportunity to explore the physiology of a facultative FeOB and constrain the genes specific to Fe(II) oxidation. We compared the growth of S. lithotrophicus ES-1 on Fe(II), thiosulfate, and both substrates together. While initial growth rates were similar, thiosulfate-grown cultures had higher yield with or without Fe(II) present, which may give ES-1 an advantage over obligate FeOB. To investigate the Fe(II) and S oxidation pathways, we conducted transcriptomics experiments, validated with reverse transcription-quantitative PCR (RT-qPCR). We explored the long-term gene expression response at different growth phases (over days to a week) and expression changes during a short-term switch from thiosulfate to Fe(II) (90 min). The dsr and sox sulfur oxidation genes were upregulated in thiosulfate cultures. The Fe(II) oxidase gene cyc2 was among the top expressed genes during both Fe(II) and thiosulfate oxidation, and addition of Fe(II) to thiosulfate-grown cells caused an increase in cyc2 expression. These results support the role of Cyc2 as the Fe(II) oxidase and suggest that ES-1 maintains readiness to oxidize Fe(II), even in the absence of Fe(II). We used gene expression profiles to further constrain the ES-1 Fe(II) oxidation pathway. Notably, among the most highly upregulated genes during Fe(II) oxidation were genes for alternative complex III, reverse electron transport, and carbon fixation. This implies a direct connection between Fe(II) oxidation and carbon fixation, suggesting that CO2 is an important electron sink for Fe(II) oxidation. IMPORTANCE Neutrophilic FeOB are increasingly observed in various environments, but knowledge of their ecophysiology and Fe(II) oxidation mechanisms is still relatively limited. Sideroxydans isolates are widely observed in aquifers, wetlands, and sediments, and genome analysis suggests metabolic flexibility contributes to their success. The type strain ES-1 is unusual among neutrophilic FeOB isolates, as it can grow on either Fe(II) or a non-Fe(II) substrate, thiosulfate. Almost all our knowledge of neutrophilic Fe(II) oxidation pathways comes from genome analyses, with some work on metatranscriptomes. This study used culture-based experiments to test the genes specific to Fe(II) oxidation in a facultative FeOB and refine our model of the Fe(II) oxidation pathway. We gained insight into how facultative FeOB like ES-1 connect Fe, S, and C biogeochemical cycling in the environment and suggest a multigene indicator would improve understanding of Fe(II) oxidation activity in environments with facultative FeOB.

Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments.

BACKGROUND: The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. RESULTS: We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. CONCLUSION: Taken together, we discovered that Fgf9 may play an influential role in muscle-bone cross-talk during embryonic and postnatal development.

Urbanization pressures alter tree rhizosphere microbiomes.

The soil microbial community (SMC) provides critical ecosystem services including organic matter decomposition, soil structural formation, and nutrient cycling. Studies suggest plants, specifically trees, act as soil keystone species controlling SMC structure via multiple mechanisms (e.g., litter chemistry, root exudates, and canopy alteration of precipitation). Tree influence on SMC is shaped by local/regional climate effects on forested environments and the connection of forests to surrounding landscapes (e.g., urbanization). Urban soils offer an ideal analog to assess the influence of environmental conditions versus plant species-specific controls on SMC. We used next generation high throughput sequencing to characterize the SMC of specific tree species (Fagus grandifolia [beech] vs Liriodendron tulipifera [yellow poplar]) across an urban-rural gradient. Results indicate SMC dissimilarity within rural forests suggests the SMC is unique to individual tree species. However, greater urbanization pressure increased SMC similarity between tree species. Relative abundance, species richness, and evenness suggest that increases in similarity within urban forests is not the result of biodiversity loss, but rather due to greater overlap of shared taxa. Evaluation of soil chemistry across the rural-urban gradient indicate pH, Ca+, and organic matter are largely responsible for driving relative abundance of specific SMC members.

k-mer-Based Metagenomics Tools Provide a Fast and Sensitive Approach for the Detection of Viral Contaminants in Biopharmaceutical and Vaccine Manufacturing Applications Using Next-Generation Sequencing.

Adventitious agent detection during the production of vaccines and biotechnology-based medicines is of critical importance to ensure the final product is free from any possible viral contamination. Increasing the speed and accuracy of viral detection is beneficial as a means to accelerate development timelines and to ensure patient safety. Here, several rapid viral metagenomics approaches were tested on simulated next-generation sequencing (NGS) data sets and existing data sets from virus spike-in studies done in CHO-K1 and HeLa cell lines. It was observed that these rapid methods had comparable sensitivity to full-read alignment methods used for NGS viral detection for these data sets, but their specificity could be improved. A method that first filters host reads using KrakenUniq and then selects the virus classification tool based on the number of remaining reads is suggested as the preferred approach among those tested to detect nonlatent and nonendogenous viruses. Such an approach shows reasonable sensitivity and specificity for the data sets examined and requires less time and memory as full-read alignment methods.IMPORTANCE Next-generation sequencing (NGS) has been proposed as a complementary method to detect adventitious viruses in the production of biotherapeutics and vaccines to current in vivo and in vitro methods. Before NGS can be established in industry as a main viral detection technology, further investigation into the various aspects of bioinformatics analyses required to identify and classify viral NGS reads is needed. In this study, the ability of rapid metagenomics tools to detect viruses in biopharmaceutical relevant samples is tested and compared to recommend an efficient approach. The results showed that KrakenUniq can quickly and accurately filter host sequences and classify viral reads and had comparable sensitivity and specificity to slower full read alignment approaches, such as BLASTn, for the data sets examined.

Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization, and Remediation of Larval Oyster Mortalities.

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortality rates in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family, and two were members of the Siphoviridae In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% 6 days after challenge with lethal doses of V. coralliilyticus Larval survival depended on the oyster species, the quantities of phages and vibrios applied, and the species and strain of Vibrio A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phages vB_VcorM-GR7B, vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A, and 28A, respectively). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by >90% (P < 0.0001) over 6 days with phage treatment compared to those of untreated controls. Genomic sequencing of phages 7B, 11A, and 28A revealed 207,758-, 194,800-, and 154,046-bp linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries.IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of hatcheries and the commercial shellfish industry. Shellfish pathogens like Vibrio coralliilyticus and Vibrio tubiashii have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and from both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach for stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

Investigation of allele specific expression in various tissues of broiler chickens using the detection tool VADT.

Differential abundance of allelic transcripts in a diploid organism, commonly referred to as allele specific expression (ASE), is a biologically significant phenomenon and can be examined using single nucleotide polymorphisms (SNPs) from RNA-seq. Quantifying ASE aids in our ability to identify and understand cis-regulatory mechanisms that influence gene expression, and thereby assist in identifying causal mutations. This study examines ASE in breast muscle, abdominal fat, and liver of commercial broiler chickens using variants called from a large sub-set of the samples (n = 68). ASE analysis was performed using a custom software called VCF ASE Detection Tool (VADT), which detects ASE of biallelic SNPs using a binomial test. On average ~ 174,000 SNPs in each tissue passed our filtering criteria and were considered informative, of which ~ 24,000 (~ 14%) showed ASE. Of all ASE SNPs, only 3.7% exhibited ASE in all three tissues, with ~ 83% showing ASE specific to a single tissue. When ASE genes (genes containing ASE SNPs) were compared between tissues, the overlap among all three tissues increased to 20.1%. Our results indicate that ASE genes show tissue-specific enrichment patterns, but all three tissues showed enrichment for pathways involved in translation.

Aerobic and anaerobic iron oxidizers together drive denitrification and carbon cycling at marine iron-rich hydrothermal vents.

In principle, iron oxidation can fuel significant primary productivity and nutrient cycling in dark environments such as the deep sea. However, we have an extremely limited understanding of the ecology of iron-based ecosystems, and thus the linkages between iron oxidation, carbon cycling, and nitrate reduction. Here we investigate iron microbial mats from hydrothermal vents at Lo'ihi Seamount, Hawai'i, using genome-resolved metagenomics and metatranscriptomics to reconstruct potential microbial roles and interactions. Our results show that the aerobic iron-oxidizing Zetaproteobacteria are the primary producers, concentrated at the oxic mat surface. Their fixed carbon supports heterotrophs deeper in the mat, notably the second most abundant organism, Candidatus Ferristratum sp. (uncultivated gen. nov.) from the uncharacterized DTB120 phylum. Candidatus Ferristratum sp., described using nine high-quality metagenome-assembled genomes with similar distributions of genes, expressed nitrate reduction genes narGH and the iron oxidation gene cyc2 in situ and in response to Fe(II) in a shipboard incubation, suggesting it is an anaerobic nitrate-reducing iron oxidizer. Candidatus Ferristratum sp. lacks a full denitrification pathway, relying on Zetaproteobacteria to remove intermediates like nitrite. Thus, at Lo'ihi, anaerobic iron oxidizers coexist with and are dependent on aerobic iron oxidizers. In total, our work shows how key community members work together to connect iron oxidation with carbon and nitrogen cycling, thus driving the biogeochemistry of exported fluids.

Understanding ER+ Breast Cancer Dormancy Using Bioinspired Synthetic Matrices for Long-Term 3D Culture and Insights into Late Recurrence.

Late recurrences of breast cancer are hypothesized to originate from disseminated tumor cells that re-activate after a long period of dormancy, ≥5 years for estrogen-receptor positive (ER+) tumors. An outstanding question remains as to what the key microenvironment interactions are that regulate this complex process, and well-defined human model systems are needed for probing this. Here, a robust, bioinspired 3D ER+ dormancy culture model is established and utilized to probe the effects of matrix properties for common sites of late recurrence on breast cancer cell dormancy. Formation of dormant micrometastases over several weeks is examined for ER+ cells (T47D, BT474), where the timing of entry into dormancy versus persistent growth depends on matrix composition and cell type. In contrast, triple negative cells (MDA-MB-231), associated with early recurrence, are not observed to undergo long-term dormancy. Bioinformatic analyses quantitatively support an increased "dormancy score" gene signature for ER+ cells (T47D) and reveal differential expression of genes associated with different biological processes based on matrix composition. Further, these analyses support a link between dormancy and autophagy, a potential survival mechanism. This robust model system will allow systematic investigations of other cell-microenvironment interactions in dormancy and evaluation of therapeutics for preventing late recurrence.

The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology.

Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7-/- mice. Early postnatal Tdrd7-/- animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7-/- lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7-/- cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7-/- lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7-/- cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7-/- lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7-/- fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7-ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7's novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.

Iroki: automatic customization and visualization of phylogenetic trees.

Phylogenetic trees are an important analytical tool for evaluating community diversity and evolutionary history. In the case of microorganisms, the decreasing cost of sequencing has enabled researchers to generate ever-larger sequence datasets, which in turn have begun to fill gaps in the evolutionary history of microbial groups. However, phylogenetic analyses of these types of datasets create complex trees that can be challenging to interpret. Scientific inferences made by visual inspection of phylogenetic trees can be simplified and enhanced by customizing various parts of the tree. Yet, manual customization is time-consuming and error prone, and programs designed to assist in batch tree customization often require programming experience or complicated file formats for annotation. Iroki, a user-friendly web interface for tree visualization, addresses these issues by providing automatic customization of large trees based on metadata contained in tab-separated text files. Iroki's utility for exploring biological and ecological trends in sequencing data was demonstrated through a variety of microbial ecology applications in which trees with hundreds to thousands of leaf nodes were customized according to extensive collections of metadata. The Iroki web application and documentation are available at https://www.iroki.net or through the VIROME portal http://virome.dbi.udel.edu. Iroki's source code is released under the MIT license and is available at https://github.com/mooreryan/iroki.