Trojan horses in the marine realm: characterizing protistan parasite ecology in coastal waters

Protists are taxonomically and metabolically diverse drivers of energy and nutrient flow in the marine environment, with recent research suggesting significant roles in global carbon cycling throughout the water column. Top-down controls on planktonic protists include grazing and parasitism, processes that both contribute to nutrient transfer and biogeochemical cycling in the global ocean. Recent global surveys of eukaryotic small subunit ribosomal RNA molecular signatures have highlighted the fact that parasites belonging to the marine alveolate order Syndiniales are both abundant and ubiquitous in coastal and open ocean environments, suggesting a major role for this taxon in marine food webs. Two coastal sites, Saanich Inlet (Vancouver Island, BC) and Salt Pond (Falmouth, MA, USA) were selected as model ecosystems to examine the impacts of Syndinian parasitism on protist communities. Data presented in this thesis combines high-resolution sampling, water chemistry (including nutrients) analyses, molecular marker gene analyses, fluorescence in situ hybridization, and modeling to address key knowledge gaps regarding syndinian ecology. Information is presented on previously undescribed putative host taxa, the prevalence of syndinian parasites and infections on different hosts in coastal waters, and a framework for modeling host-parasite interactions based on field observations.

Variability in Host Specificity and Functional Potential of Antarctic Sponge-Associated Bacterial Communities

Sponge-associated microorganisms are essential for sponge survival. They play an important role in recycling nutrients and, therefore, in the maintenance of the ecosystem. These microorganisms are diverse, species-specific, and different from those in the surrounding seawater. Bacterial sponge symbionts have been extensively studied in the tropics; however, little is known about these microorganisms in sponges from high-latitude environments. Sponges can cover up to 80% of the benthos in Antarctica and are crucial architects for the marine food web. In this study, we present analyses of the bacterial symbionts of three sponges: Haliclona (Rhizoniera) sp., Hymeniacidon torquata, and Isodictya kerguelenensis from the Western Antarctic Peninsula (WAP) with the aim to determine variations on the specificity of the bacteria–sponge interactions and potential signatures on their predicted functional profiles. We use high-throughput 16S rRNA gene sequencing of 30 sponge individuals inhabiting South Bay (Palmer Archipelago, WAP) to describe their microbiome taxonomy and diversity and predict potential functional profiles based on this marker gene. Our work shows similar bacterial community composition profiles among the same sponge species, although the symbiotic relationship is not equally conserved among the three Antarctic sponges. The number of species-specific core operational taxonomic units (OTUs) of these Antarctic sponges was low, with important differences between the total abundance accounted for these OTUs. Only eight OTUs were shared between the three sponge species. Analyses of the functional potential revealed that despite the high host–symbiont specificity, the inferred functions are conserved among these microbiomes, although with differences in the abundance of specific functions. H. torquata showed the highest level of intra-specificity and a higher potential of pathways related to energy metabolism, metabolisms of terpenoids and polyketides, and biosynthesis of other secondary metabolites. Overall, this work shows variations in the specificity of the sponge-associated bacterial communities, differences in how hosts and symbionts establish their relations, and in their potential functional capabilities.

Genome Sequence of a Thermoacidophilic Methanotroph Belonging to the Verrucomicrobiota Phylum from Geothermal Hot Springs in Yellowstone National Park: A Metagenomic Assembly and Reconstruction

Verrucomicrobiotal methanotrophs are thermoacidophilic methane oxidizers that have been isolated from volcanic and geothermal regions of the world. We used a metagenomic approach that entailed obtaining the whole genome sequence of a verrucomicrobiotal methanotroph from a microbial consortium enriched from samples obtained from Nymph Lake (89.9 °C, pH 2.73) in Yellowstone National Park in the USA. To identify and reconstruct the verrucomicrobiotal genome from Illumina NovaSeq 6000 sequencing data, we constructed a bioinformatic pipeline with various combinations of de novo assembly, alignment, and binning algorithms. Based on the marker gene (pmoA), we identified and assembled the Candidatus Methylacidiphilum sp. YNP IV genome (2.47 Mbp, 2392 ORF, and 41.26% GC content). In a comparison of average nucleotide identity between Ca. Methylacidiphilum sp. YNP IV and Ca. Methylacidiphilum fumariolicum SolV, its closest 16S rRNA gene sequence relative, is lower than 95%, suggesting that Ca. Methylacidiphilum sp. YNP IV can be regarded as a different species. The Ca. Methylacidiphilum sp. YNP IV genome assembly showed most of the key genes for methane metabolism, the CBB pathway for CO2 fixation, nitrogen fixation and assimilation, hydrogenases, and rare earth elements transporter, as well as defense mechanisms. The assembly and reconstruction of a thermoacidophilic methanotroph belonging to the Verrucomicrobiota phylum from a geothermal environment adds further evidence and knowledge concerning the diversity of biological methane oxidation and on the adaptation of this geochemically relevant reaction in extreme environments.

EARLY FLOWERING 3 and Photoperiod Sensing in Brachypodium distachyon

The proper timing of flowering, which is key to maximize reproductive success and yield, relies in many plant species on the coordination between environmental cues and endogenous developmental programs. The perception of changes in day length is one of the most reliable cues of seasonal change, and this involves the interplay between the sensing of light signals and the circadian clock. Here, we describe a Brachypodium distachyon mutant allele of the evening complex protein EARLY FLOWERING 3 (ELF3). We show that the elf3 mutant flowers more rapidly than wild type plants in short days as well as under longer photoperiods but, in very long (20 h) days, flowering is equally rapid in elf3 and wild type. Furthermore, flowering in the elf3 mutant is still sensitive to vernalization, but not to ambient temperature changes. Molecular analyses revealed that the expression of a short-day marker gene is suppressed in elf3 grown in short days, and the expression patterns of clock genes and flowering time regulators are altered. We also explored the mechanisms of photoperiodic perception in temperate grasses by exposing B. distachyon plants grown under a 12 h photoperiod to a daily night break consisting of a mixture of red and far-red light. We showed that 2 h breaks are sufficient to accelerate flowering in B. distachyon under non-inductive photoperiods and that this acceleration of flowering is mediated by red light. Finally, we discuss advances and perspectives for research on the perception of photoperiod in temperate grasses.

ASAP 2: a pipeline and web server to analyze marker gene amplicon sequencing data automatically and consistently

Background Amplicon sequencing of marker genes such as 16S rDNA have been widely used to survey and characterize microbial community. However, the complex data analyses have required many interfering manual steps often leading to inconsistencies in results. Results Here, we have developed a pipeline, amplicon sequence analysis pipeline 2 (ASAP 2), to automate and glide through the processes without the usual manual inspections and user’s interference, for instance, in the detection of barcode orientation, selection of high-quality region of reads, and determination of resampling depth and many more. The pipeline integrates all the analytical processes such as importing data, demultiplexing, summarizing read profiles, trimming quality, denoising, removing chimeric sequences and making the feature table among others. The pipeline accepts multiple file formats as input including multiplexed or demultiplexed, paired-end or single-end, barcode inside or outside and raw or intermediate data (e.g. feature table). The outputs include taxonomic classification, alpha/beta diversity, community composition, ordination analysis and statistical tests. ASAP 2 supports merging multiple sequencing runs which helps integrate and compare data from different sources (public databases and collaborators). Conclusions Our pipeline minimizes hands-on interference and runs amplicon sequence variant (ASV)-based amplicon sequencing analysis automatically and consistently. Our web server assists researchers that have no access to high performance computer (HPC) or have limited bioinformatics skills. The pipeline and web server can be accessed at https://github.com/tianrenmaogithub/asap2 and https://hts.iit.edu/asap2, respectively.

MiR-27a-3p Targets GLP1R to Regulate Differentiation, Autophagy, and Release of Inflammatory Factors in Pre-Osteoblasts via the AMPK Signaling Pathway

Objective: Osteoporosis is caused by the dysregulation of bone homeostasis which is synergistically mediated by osteoclasts and osteoblasts. MiR-27a-3p is a key inhibitor of bone formation. Hence, unearthing the downstream target gene of miR-27a-3p is of great significance to understand the molecular mechanism of osteoporosis.Methods: Bioinformatics analysis was utilized to find the downstream target gene of miR-27a-3p, and dual-luciferase reporter assay was conducted to validate the interplay of miR-27a-3p and GLP1R. Besides, qRT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) were employed to verify the impact of miR-27a-3p on GLP1R expression and the differentiation, autophagy, and inflammatory response of MC3T3-E1 pre-osteoblasts.Results: Dual-luciferase assay validated that miR-27a-3p directly targeted GLP1R. Additionally, posttreatment of MC3T3-E1 cells with miR-27a-3p mimics resulted in a remarkable decrease in expression levels of GLP1R, cell differentiation marker gene, autophagy marker gene, and AMPK. These results indicated that miR-27a-3p targeted GLP1R to inhibit AMPK signal activation and pre-osteoblast differentiation and autophagy, while promoting the release of inflammatory factors.Conclusion: The miR-27a-3p/GLP1R regulatory axis in pre-osteoblasts contributes to understanding the molecular mechanism of osteoporosis.

Baf45a Mediated Chromatin Remodeling Promotes Transcriptional Activation for Osteogenesis and Odontogenesis

Chromatin remodeling, specifically the tissue-specific regulation in mineralized tissues, is an understudied avenue of gene regulation. Here we show that Baf45a and Baf45d, two Baf45 homologs belong to ATPase-dependent SWI/SNF chromatin remodeling complex, preferentially expressed in osteoblasts and odontoblasts compared to Baf45b and Baf45c. Recently, biochemical studies revealed that BAF45A associates with Polybromo-associated BAF (PBAF) complex. However, the BAF45D subunit belongs to the polymorphic canonical BRG1-associated factor (cBAF) complex. Protein profiles of osteoblast and odontoblast differentiation uncovered a significant increase of BAF45A and PBAF subunits during early osteoblast and odontoblast maturation. Chromatin immunoprecipitation sequencing (ChIP-seq) during the bone marrow stromal cells (BMSCs) differentiation showed higher histone H3K9 and H3K27 acetylation modifications in the promoter of Baf45a and Baf45d and increased binding of bone and tooth specific transcription factor RUNX2. Overexpression of Baf45a in osteoblasts activates genes essential for the progression of osteoblast maturation and mineralization. Furthermore, shRNA-mediated knockdown of Baf45a in odontoblasts leads to markedly altered genes responsible for the proliferation, apoptosis, DNA repair, and modest decrease in dentinogenic marker gene expression. Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) assay in Baf45a knockout osteoblasts revealed a noticeable reduction in chromatin accessibility of osteoblast and odontoblast specific genes, along with transcription factor Atf4 and Klf4. Craniofacial mesenchyme-specific loss of Baf45a modestly reduced the mineralization of the tooth and mandibular bone. These findings indicated that BAF45A-dependent mineralized tissue-specific chromatin remodeling through PBAF-RUNX2 crosstalk results in transcriptional activation is critical for early differentiation and matrix maturation of mineralized tissues.

TaxaTarget: Fast, Sensitive, and Precise Classification of Microeukaryotes in Metagenomic Data

BackgroundMicrobial eukaryotes are nearly ubiquitous in microbiomes on Earth and contribute to many integral ecological functions. Metagenomics is a proven tool for studying the microbial diversity, functions, and ecology of microbiomes, but has been underutilized for microeukaryotes due to the computational challenges they present. For taxonomic classification, the use of a eukaryotic marker gene database can improve the computational efficiency, precision and sensitivity. However, state-of-the-art tools which use marker gene databases implement universal thresholds for classification rather than dynamically learning the thresholds from the database structure, impacting the accuracy of the classification process.ResultsHere we introduce taxaTarget, a method for the taxonomic classification of microeukaryotes in metagenomic data. Using a database of eukaryotic marker genes and a supervised learning approach for training, we learned the discriminatory power and classification thresholds for each 20 amino acid region of each marker gene in our database. This approach provided improved sensitivity and precision compared to other state-of-the-art approaches, with rapid runtimes and low memory usage. Additionally, taxaTarget was better able to detect the presence of multiple closely related species as well as species with no representative sequences in the database. One of the greatest challenges faced during the development of taxaTarget was the general sparsity of available sequences for microeukaryotes. Several algorithms were implemented, including threshold padding, which effectively handled the missing training data and reduced classification errors. Using taxaTarget on metagenomes from human fecal microbiomes, a broader range of genera were detected, including multiple parasites that the other tested tools missed.ConclusionData-driven methods for learning classification thresholds from the structure of an input database can provide granular information about the discriminatory power of the sequences and improve the sensitivity and precision of classification. These methods will help facilitate a more comprehensive analysis of metagenomic data and expand our knowledge about the diverse eukaryotes in microbial communities.

Single-cell analysis of microglial transcriptomic diversity in subarachnoid hemorrhage

Background: Subarachnoid hemorrhage (SAH) is a severe stroke and the advanced treatment for SAH is still limited. Recent studies have shown that microglia-mediated neuroinflammation plays a critical role in the pathogenesis of SAH. Microglia can transform their states in response to central nervous system injury. However, the transcriptomic features of microglia remained unknown in SAH. Recent developed single-cell RNA sequencing (scRNA-seq) provides a possible way to solve this problem. Methods: Endovascular perforation (EVP) murine SAH model was established to reproduce experimental SAH. Microglia states are examined with immune staining and quantitate analysis. Post-SAH microglial single-cell suspension were harvest and sequenced using 10X scRNA-seq platform. Then, the detailed single-cell transcriptomic characterization of post-SAH microglia were analyzed with bioinformatics. Results: Transcriptional analysis revealed at least ten diverse microglial subgroups, including SAH-associated microglia (SAM), inflammatory-associated microglia (IAM) and proliferation-associated microglia (PAM), which all exhibit distinct marker gene expression patterns. Microglia subsets interaction reveals the functional relationship between elevated signaling pathways and microglial sub-populations in SAH. Receptor-ligand pair analysis revealed that complex inter-cellular interactions exist between the microglia subsets and other cell types, and indicated that microglia are important mediators of neuroinflammation after SAH. Integrated analysis with normal microglia further proved the existence of these microglia subpopulations and different gene markers associated with SAH were clarified. Conclusions: Collectively, we first report the single-cell transcriptome of post-SAH microglia and found specific biomarkers related to the neuroinflammation in SAH. These results enhanced our understanding of the pathological mechanisms of microglial response to SAH, and may guide future development of SAH monitoring methods and therapeutics.

Screening and Functional Verification of Selectable Marker Genes for Cordyceps militaris

The selectable marker genes are necessary resistance genes for gene knockout, gene complementation, and gene overexpression in filamentous fungi. Moreover, the more sensitive the filamentous fungi are to antibiotics, the more helpful it is to screen the target transformants. In order to obtain the antibiotic (or herbicide) which can effectively inhibit the growth of Cordyceps militaris and verify the function of the corresponding resistance gene in C. militaris, the sensitivity of C. militaris to hygromycin and glufosinate ammonium was compared to determine the resistance gene that was more suitable for the screening of C. militaris transformants. The binary vector of the selectable marker gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the selectable marker gene in C. militaris was verified by the Agrobacterium tumefaciens-mediated transformation method. The results showed that C. militaris was more sensitive to glufosinate ammonium than hygromycin. The growth of C. militaris could be completely inhibited by 250 μg/mL glufosinate ammonium. The expression cassette of the glufosinate ammonium resistance gene (bar gene) was successfully constructed by DJ-PCR. The binary vector pCAMBIA0390-Bar was successfully constructed by homologous recombination. The bar gene of the vector pCAMBIA0390-Bar was successfully integrated into the C. militaris genome and could be highly expressed in the transformants of C. militaris. This study will promote the identification of C. militaris gene function and reveal the biosynthetic pathways of bioactive components in C. militaris.