Comparative Genome Analysis of the Genus Thiothrix Involving Three Novel Species, Thiothrix subterranea sp. nov. Ku-5, Thiothrix litoralis sp. nov. AS and “Candidatus Thiothrix anitrata” sp. nov. A52, Revealed the Conservation of the Pathways of Dissimilatory Sulfur Metabolism and Variations in the Genetic Inventory for Nitrogen Metabolism and Autotrophic Carbon Fixation

Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus Thiothrix. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus Thiothrix with the proposed names Thiothrix subterranea sp. nov. Ku-5T, Thiothrix litoralis sp. nov. AST, and “Candidatus Thiothrix anitrata” sp. nov. A52. The complete genome sequences of Thiothrix fructosivorans QT and Thiothrix unzii A1T were determined. Complete genomes of seven Thiothrix isolates, as well as two MAGs, were used for pangenome analysis. The Thiothrix core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (SoxAXBYZ), direct (soeABC) and indirect (aprAB, sat) pathways of sulfite oxidation, sulfur oxidation complex Dsr (dsrABEFHCEMKLJONR), sulfide oxidation systems SQR (sqrA, sqrF), and FCSD (fccAB) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.

Abstract P227: Gq Signaling In The Placental Syncytiotrophoblast Layer During Preeclampsia

Hormones implicated in preeclampsia (PE) such as angiotensin, endothelin, and vasopressin signal via receptors coupled to the Gq cascade, and Regulator of G protein Signaling-2 (RGS2) buffers this signaling. We have published that RGS2 expression is decreased in human PE placenta, and reducing RGS2 in placenta causes development of key features of PE in mice. New in situ hybridization data indicate that in both humans and mice, RGS2 is abundant among many cell types in the placenta, including the syncytiotrophoblast (STB) layer. In addition, RGS2 expression in the human STB layer is reduced during PE. As this layer is strongly implicated in PE, these data lead us to hypothesize a critical Gq-buffering role for RGS2 in STB cells to prevent PE. To explore the effect of excess Gq signaling within the STB layer, we utilized a Cre-Lox approach to cause expression of the Gq-coupled hM3Dq DREADD throughout the fetoplacental unit (dam: hM3Dq+, sire: Actb-Cre+) or only within the STB layer (dam: hM3Dq+, sire: Gcm1-Cre+), and then activated the hM3Dq receptor via clozapine N-oxide (CNO, 0.5 to 2 mg/kg) injection in mid-gestation (GD12.5-14.5) before tissue collection at GD14.5. Gαq activation throughout the fetoplacental unit (Actb-Cre model) severely restricted fetoplacental growth compared to saline-injected controls (n=2 vs 3; placenta: 0.027±0.006 vs 0.115±0.021 g; p<0.05, and fetus: 0.048±0.007 vs 0.268±0.010 g; p<0.05). Similarly, placentas expressing hM3Dq only in STB cells (Gcm1-Cre model) had reduced placental (n=3 0.116±0.022 vs 0.201±0.036 g; p=0.05) and possibly fetal (n=3 0.1112±0.036 vs 0.247±0.028 g; p=0.06) masses after CNO. Vascularization (assessed by CD31 immunostain) was disproportionately reduced in the labyrinth layer of the Actb-Cre model after CNO (n=2 vs 3; 20.189±3.382 vs 35.762±1.976 % area; p<0.05), despite no relative change in layer (ie, decidua/junctional zone/labyrinth) thicknesses. Preliminary results indicate similar findings in the Gcm1-Cre model (n=1 17 vs 25 % area). These data highlight the pathological consequence of excess Gq signaling in the STB layer. Ongoing studies are aimed at characterizing maternal phenotypes in these models and the consequence of STB-specific deletion of RGS2 upon sensitivity to Gq stimulators.

Disruption of Brachypodium Lichenase Alters Metabolism of Mixed-linkage Glucan and Starch

Mixed-linkage glucan (MLG), which is widely distributed in grasses, is a polysaccharide highly abundant in cell walls of grass endosperm and young vegetative tissues. Lichenases are enzymes that hydrolyze MLG first identified in MLG-rich lichens. In this study, we identify a gene encoding a lichenase we name Brachypodium distachyon LICHENASE 1 (BdLCH1), which is highly expressed in the endosperm of germinating seeds and coleoptiles and at lower amounts in mature shoots. RNA in situ hybridization showed that BdLCH1 is primarily expressed in chlorenchyma cells of mature leaves and internodes. Disruption of BdLCH1 resulted in an eight-fold increase in MLG content in senesced leaves. Consistent with the in situ hybridization data, immunolocalization results showed that MLG was not removed in chlorenchyma cells of lch1 mutants as it was in wild type and implicate the BdLCH1 enzyme in removing MLG in chlorenchyma cells in mature vegetative tissues. We also show that MLG accumulation in lch1 mutants was resistant to dark induced degradation, and eight-week-old lch1 plants showed a faster rate of starch breakdown than wild type in darkness. Our results suggest a role for BdLCH1 in modifying the cell wall to support highly metabolically active cells.

Motilimonas cestriensis sp. nov., isolated from an inland brine spring in Northern England

A novel slightly halophilic Gram-stain-negative bacterial strain (MKS20T) was isolated from a brine sample collected from one of the Anderton brine springs in the Cheshire salt district, located in Northern England. Phylogenetic analysis of the 16S rRNA gene sequence revealed a close proximity to Motilimonas eburnea (98.30 %), followed by Motilimonas pumila (96.62 %), the two currently described species within the genus Motilimonas . Strain MKS20T forms white-beige-pigmented colonies and grows optimally at 28–30 °C, in 1–3 % (w/v) NaCl and at pH 7–7.5. The strain was facultatively anaerobic and showed a broader range of carbohydrate use than other species in the genus Motilimonas . Q-8 was the sole respiratory quinone and the major fatty acids (>10 %) were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and C16 : 0. The polar lipid profile included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyglycerol and several unidentified lipids. The G+C content of the genomic DNA was 44.2 mol%. Average nucleotide identity and DNA–DNA hybridization data were consistent with assignment to a separate species. Based on the phylogenetic and genomic-based analyses, as well as physiological and biochemical characteristics, we propose that strain MKS20T (=DSM 109936T, MCCC 1K04071T) represents a new species of the genus Motilimonas , with the name Motilimonas cestriensis sp. nov.

Homologous laminar organization of the mouse and human subiculum

The subiculum is the major output component of the hippocampal formation and one of the major brain structures most affected by Alzheimer’s disease. Our previous work revealed a hidden laminar architecture within the mouse subiculum. However, the rotation of the hippocampal longitudinal axis across species makes it unclear how the laminar organization is represented in human subiculum. Using in situ hybridization data from the Allen Human Brain Atlas, we demonstrate that the human subiculum also contains complementary laminar gene expression patterns similar to the mouse. In addition, we provide evidence that the molecular domain boundaries in human subiculum correspond to microstructural differences observed in high resolution MRI and fiber density imaging. Finally, we show both similarities and differences in the gene expression profile of subiculum pyramidal cells within homologous lamina. Overall, we present a new 3D model of the anatomical organization of human subiculum and its evolution from the mouse.

Mechanisms Generating Cancer Genome Complexity: Back to the Future

Understanding the mechanisms underlying cancer genome evolution has been a major goal for decades. A recent study combining live cell imaging and single-cell genome sequencing suggested that interwoven chromosome breakage-fusion-bridge cycles, micronucleation events and chromothripsis episodes drive cancer genome evolution. Here, I discuss the “interphase breakage model,” suggested from prior fluorescent in situ hybridization data that led to a similar conclusion. In this model, the rapid genome evolution observed at early stages of gene amplification was proposed to result from the interweaving of an amplification mechanism (breakage-fusion-bridge cycles) and of a deletion mechanism (micronucleation and stitching of DNA fragments retained in the nucleus).

Marmoset Brain ISH Data Revealed Molecular Difference Between Cortical Folding Patterns

Literature studies have demonstrated the structural, connectional, and functional differences between cortical folding patterns in mammalian brains, such as convex and concave patterns. However, the molecular underpinning of such convex/concave differences remains largely unknown. Thanks to public access to a recently released set of marmoset whole-brain in situ hybridization data by RIKEN, Japan; this data’s accessibility empowers us to improve our understanding of the organization, regulation, and function of genes and their relation to macroscale metrics of brains. In this work, magnetic resonance imaging and diffusion tensor imaging macroscale neuroimaging data in this dataset were used to delineate convex/concave patterns in marmoset and to examine their structural features. Machine learning and visualization tools were employed to investigate the possible transcriptome difference between cortical convex and concave patterns. Experimental results demonstrated that a collection of genes is differentially expressed in convex and concave patterns, and their expression profiles can robustly characterize and differentiate the two folding patterns. More importantly, neuroscientific interpretations of these differentially expressed genes, as well as axonal guidance pathway analysis and gene enrichment analysis, offer novel understanding of structural and functional differences between cortical folding patterns in different regions from a molecular perspective.

The Nucleome Data Bank: web-based resources to simulate and analyze the three-dimensional genome

We introduce the Nucleome Data Bank (NDB), a web-based platform to simulate and analyze the three-dimensional (3D) organization of genomes. The NDB enables physics-based simulation of chromosomal structural dynamics through the MEGABASE + MiChroM computational pipeline. The input of the pipeline consists of epigenetic information sourced from the Encode database; the output consists of the trajectories of chromosomal motions that accurately predict Hi-C and fluorescence insitu hybridization data, as well as multiple observations of chromosomal dynamics in vivo. As an intermediate step, users can also generate chromosomal sub-compartment annotations directly from the same epigenetic input, without the use of any DNA–DNA proximity ligation data. Additionally, the NDB freely hosts both experimental and computational structural genomics data. Besides being able to perform their own genome simulations and download the hosted data, users can also analyze and visualize the same data through custom-designed web-based tools. In particular, the one-dimensional genetic and epigenetic data can be overlaid onto accurate 3D structures of chromosomes, to study the spatial distribution of genetic and epigenetic features. The NDB aims to be a shared resource to biologists, biophysicists and all genome scientists. The NDB is available at https://ndb.rice.edu.

Gene selection for optimal prediction of cell position in tissues from single-cell transcriptomics data

Single-cell RNA-sequencing (scRNAseq) technologies are rapidly evolving. Although very informative, in standard scRNAseq experiments, the spatial organization of the cells in the tissue of origin is lost. Conversely, spatial RNA-seq technologies designed to maintain cell localization have limited throughput and gene coverage. Mapping scRNAseq to genes with spatial information increases coverage while providing spatial location. However, methods to perform such mapping have not yet been benchmarked. To fill this gap, we organized the DREAM Single-Cell Transcriptomics challenge focused on the spatial reconstruction of cells from the Drosophila embryo from scRNAseq data, leveraging as silver standard, genes with in situ hybridization data from the Berkeley Drosophila Transcription Network Project reference atlas. The 34 participating teams used diverse algorithms for gene selection and location prediction, while being able to correctly localize clusters of cells. Selection of predictor genes was essential for this task. Predictor genes showed a relatively high expression entropy, high spatial clustering and included prominent developmental genes such as gap and pair-rule genes and tissue markers. Application of the top 10 methods to a zebra fish embryo dataset yielded similar performance and statistical properties of the selected genes than in the Drosophila data. This suggests that methods developed in this challenge are able to extract generalizable properties of genes that are useful to accurately reconstruct the spatial arrangement of cells in tissues.