Project description:Black corals, ecologically important cnidarians found from shallow to deep ocean depths, form a strong yet flexible skeleton of sclerotized chitin and other biomolecules including proteins. The structure and mechanical properties of the chitin component of the skeleton have been well-characterized. However, the protein component has remained a mystery. Here we used liquid chromatography-tandem mass spectrometry to sequence proteins extracted from two species of common Red Sea black corals following either one or two cleaning steps. We detected hundreds of proteins between the two corals, nearly 70 of which are each others’ reciprocal best BLAST hit. Unlike stony corals, only a few of the detected proteins were moderately acidic (biased toward aspartic and/or glutamic acid residues) suggesting less of a role for these types of proteins in black coral skeleton formation as compared to stony corals. No distinct chitin binding domains were found in the proteins, but proteins annotated as having a role in protein and chitin modifications were detected. Our results support the integral role of proteins in black coral skeleton formation, structure, and function.
Project description:In multi-cellular organisms, biological function emerges when cells of heterogeneous types and states are combined into complex tissues. Nevertheless unbiased dissection of tissues into coherent cell subpopulations is currently lacking. We introduce an automated, massively parallel single cell RNA sequencing method for intuitively analyzing in-vivo transcriptional states in thousands of single cells. Combined with unsupervised classification algorithms, it facilitates ab initio and marker-free characterization of classical hematopoietic cell types from splenic tissues. Importantly, modeling single cells transcriptional states in dendritic cells subpopulations, where a cell type hierarchy is difficult to define with marker-based approaches, uncovers complex combinatorial activity of multiple gene modules and capture cell-to-cell variability in steady state conditions and following pathogen activation. Massively parallel single cell RNA-seq thereby emerges as an effective tool for unbiased dissection of complex tissues. CD11c+ enriched splenocyte mRNA profiles from single cells were generated by deep sequencing of thousands of single cells, sequenced in several batches in an Illumina Hiseq 2000 The 'umitab.txt' processed data file contains the mRNA counts (post-filtering RMT counts) of a gene per each well (columns) The 'experimental_design.txt' contains a detailed information regarding each well. The 'readme0421.txt' was provided with details about each supplementary file.
Project description:Within the bone marrow, hematopoietic stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with the in vivo potential or gene regulatory mechanisms. Here we comprehensively identify myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups showing unexpected transcriptional priming towards seven differentiation fates, but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting the process is tightly regulated. Histone maps and knockout assays are consistent with the transcriptional states while traditional transplantation experiments are only partially overlapping myeloid transcriptional priming. Our analyses uncover the function of the underlying regulatory mechanisms for several sub groups and establishes a general framework for dissecting hematopoiesis. Bone marrow Lin- cKit+ Sca1- myeloid progenitors mRNA profiles from single cells were generated by deep sequencing of thousands of single cells, sequenced in several batches in an Illumina NextSeq Please note that [1] raw data files were processed as single-ended file since second read (mate) files contain only cell/molecule barcodes and therefore, not provided. This information was appended to the fastq entry header [2] The 'experimental_design.txt' file explains the correspondence of each single cell (WXXXX) in the 'umitab.txt' to a sample (ABXXXX).
Project description:We develop a new ChIpseq method (iChIP) to profile chromatin states of low cell number samples. We use IChIP to profile the chromatin dynamics during hematopoiesis across 16 different cell types which include the principal hematopoietic progenitors 3' RNA-seq for digital gene expression quantitation across multiple cell types.
Project description:Classical embryological studies revealed that during mid-embryogenesis vertebrates show similar morphologies. This âphylotypic stageâ has recently received support from transcriptome analyses, which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a âzootypicâ stage, however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum, including a segmented worm, a flatworm, a roundworm, a water bear, a fruitfly, a sea urchin, a zebrafish, a sea anemone, a sponge, and a comb jelly. For each species, we collected the embryonic transcriptomes at ~100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly, we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation, and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes, also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. 106 single embryo samples
Project description:Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress-responsive genes and transcription factors, revealed wide gene transcript reprogramming, preceded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and 40, which stimulate JA-signaling via suppression of JAZ repressors and negative-regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand, an increased colonization rate was found in roots of the FMO1 knockout mutant. Two-condition experiment: Roots treated with Trichoderma vs. Control untreated roots. Biological replicates: 2 control replicates, 2 treated replicates. 1 dye-swap.
Project description:Stony corals generate their calcium carbonate exoskeleton in a highly controlled biomineralization process mediated by a variety of macromolecules including proteins. Fully identifying and classifying these proteins is crucial to understanding their role in exoskeleton formation, yet no optimal method to extract and isolate and characterize coral skeletal proteins has been established and their complete composition remains obscure. Here, we tested four skeletal protein extraction protocols using acetone precipitation and ultrafiltration dialysis filters to present a comprehensive scleractinian coral skeletal proteome. We identified a total of 60 proteins in the coral skeleton, 44 of which were not present in previously published stony coral skeletal proteomes. Extracted protein treatment protocols carried out in this study revealed that there is no “one optimal method” and each protocol revealed a unique set of method-exclusive proteins. To better understand the general mechanism of skeletal protein transportation, we further examined the proteins’ gene ontology, transmembrane domains, and signal peptides. We found that transmembrane domain proteins and signal peptide secretion pathways, by themselves, could not explain the transportation of proteins to the skeleton . We therefore propose that proteins are transported to the skeletal via vesicles and possibly non-traditional secretion pathways.
Project description:Coral skeletons are materials composed of inorganic aragonitic fibers, proteins, sugars and lipids that are highly organized to form a solid body upon which the animals live. The skeleton contains more than 30 proteins, all of which are encoded in the animal genome and secreted during the biomineralization process. How these proteins are spatially related is unknown. Using a combination of chemical crosslinking and high-resolution tandem mass spectrometry, we identify, for the first time. the spatial interactions of the skeletal proteins within a stony coral. Our subsequent network analysis revealed several coral acid-rich proteins (CARPs) are invariably associated with carbonic anhydrase(s), alpha-collagen, cadherins and other calcium binding proteins. These interactions clearly show that protein-protein interactions in coral skeletons are highly coordinated and are key to understanding the formation and persistence of coral skeletons through time
Project description:RNA-seq in isogenic RBM10-proficient and RBM10-deficient cells derived from lung adenocarcinoma cell lines HCC827 (parental and RBM10 knockout; control siRNA and RBM10 siRNA) and NCI-H1299 (parental and RBM10 knockout).