High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have ...developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types.
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► We present CEL-Seq, a single-cell transcriptomics method using in vitro transcription ► We show that CEL-Seq is linear, sensitive, and reproducible ► CEL-Seq is highly multiplexed, allowing for the parallel examination of hundreds of cells ► We describe early C. elegans embryology at the single-cell transcriptomic level
Yanai and colleagues have developed CEL-Seq, an RNA-Seq method for assaying the transcriptome at the single-cell level. CEL-Seq works by barcoding and pooling dozens of samples before linearly amplifying mRNA using one round of in vitro transcription. We show that CEL-Seq gives highly reproducible, linear, and sensitive results. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution.
The uptake of apoptotic polymorphonuclear cells (PMN) by macrophages is critical for timely resolution of inflammation. High-burden uptake of apoptotic cells is associated with loss of phagocytosis ...in resolution phase macrophages. Here, using a transcriptomic analysis of macrophage subsets, we show that non-phagocytic resolution phase macrophages express a distinct IFN-β-related gene signature in mice. We also report elevated levels of IFN-β in peritoneal and broncho-alveolar exudates in mice during the resolution of peritonitis and pneumonia, respectively. Elimination of endogenous IFN-β impairs, whereas treatment with exogenous IFN-β enhances, bacterial clearance, PMN apoptosis, efferocytosis and macrophage reprogramming. STAT3 signalling in response to IFN-β promotes apoptosis of human PMNs. Finally, uptake of apoptotic cells promotes loss of phagocytic capacity in macrophages alongside decreased surface expression of efferocytic receptors in vivo. Collectively, these results identify IFN-β produced by resolution phase macrophages as an effector cytokine in resolving bacterial inflammation.
The moon jellyfish Aurelia aurita is a widespread scyphozoan species that forms large seasonal blooms. Here we provide the first comprehensive view of the entire complex life of the Aurelia Red Sea ...strain by employing transcriptomic profiling of each stage from planula to mature medusa.
A de novo transcriptome was assembled from Illumina RNA-Seq data generated from six stages throughout the Aurelia life cycle. Transcript expression profiling yielded clusters of annotated transcripts with functions related to each specific life-cycle stage. Free-swimming planulae were found highly enriched for functions related to cilia and microtubules, and the drastic morphogenetic process undergone by the planula while establishing the future body of the polyp may be mediated by specifically expressed Wnt ligands. Specific transcripts related to sensory functions were found in the strobila and the ephyra, whereas extracellular matrix functions were enriched in the medusa due to high expression of transcripts such as collagen, fibrillin and laminin, presumably involved in mesoglea development. The CL390-like gene, suggested to act as a strobilation hormone, was also highly expressed in the advanced strobila of the Red Sea species, and in the medusa stage we identified betaine-homocysteine methyltransferase, an enzyme that may play an important part in maintaining equilibrium of the medusa's bell. Finally, we identified the transcription factors participating in the Aurelia life-cycle and found that 70% of these 487 identified transcription factors were expressed in a developmental-stage-specific manner.
This study provides the first scyphozoan transcriptome covering the entire developmental trajectory of the life cycle of Aurelia. It highlights the importance of numerous stage-specific transcription factors in driving morphological and functional changes throughout this complex metamorphosis, and is expected to be a valuable resource to the community.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Biomineralization is the process by which living organisms use minerals to form hard structures that protect and support them. Biomineralization is believed to have evolved rapidly and independently ...in different phyla utilizing preexisting components. The mechanistic understanding of the regulatory networks that drive biomineralization and their evolution is far from clear. Sea urchin skeletogenesis is an excellent model system for studying both gene regulation and mineral uptake and deposition. The sea urchin calcite spicules are formed within a tubular cavity generated by the skeletogenic cells controlled by vascular endothelial growth factor (VEGF) signaling. The VEGF pathway is essential for biomineralization in echinoderms, while in many other phyla, across metazoans, it controls tubulogenesis and vascularization. Despite the critical role of VEGF signaling in sea urchin spiculogenesis, the downstream program it activates was largely unknown. Here we study the cellular and molecular machinery activated by the VEGF pathway during sea urchin spiculogenesis and reveal multiple parallels to the regulation of vertebrate vascularization. Human VEGF rescues sea urchin VEGF knockdown, vesicle deposition into an internal cavity plays a significant role in both systems, and sea urchin VEGF signaling activates hundreds of genes, including biomineralization and interestingly, vascularization genes. Moreover, five upstream transcription factors and three signaling genes that drive spiculogenesis are homologous to vertebrate factors that control vascularization. Overall, our findings suggest that sea urchin spiculogenesis and vertebrate vascularization diverged from a common ancestral tubulogenesis program, broadly adapted for vascularization and specifically coopted for biomineralization in the echinoderm phylum.
Monocyte-derived macrophages are readily differentiating cells that adapt their gene expression profile to environmental cues and functional needs. During the resolution of inflammation, monocytes ...initially differentiate to reparative phagocytic macrophages and later to pro-resolving non-phagocytic macrophages that produce high levels of IFNβ to boost resolutive events. Here, we performed in-depth analysis of phagocytic and non-phagocytic myeloid cells to reveal their distinct features. Unexpectedly, our analysis revealed that the non-phagocytic compartment of resolution phase myeloid cells is composed of Ly6C
F4/80
and Ly6C
F4/80
monocytic cells in addition to the previously described Ly6C
F4/80
satiated macrophages. In addition, we found that both Ly6C
monocytic cells differentiate to Ly6C
F4/80
macrophages, and their migration to the peritoneum is CCR2 dependent. Notably, satiated macrophages expressed high levels of IFNβ, whereas non-phagocytic monocytes of either phenotype did not. A transcriptomic comparison of phagocytic and non-phagocytic resolution phase F4/80
macrophages showed that both subtypes express similar gene signatures that make them distinct from other myeloid cells. Moreover, we confirmed that these macrophages express closer transcriptomes to monocytes than to resident peritoneal macrophages (RPM) and resemble resolutive Ly6C
macrophages and monocyte-derived macrophages more than their precursors, inflammatory Ly6C
monocytes, recovered following liver injury and healing, and thioglycolate-induced peritonitis, respectively. A direct comparison of these subsets indicated that the non-phagocytic transcriptome is dominated by satiated macrophages and downregulate gene clusters associated with excessive tissue repair and fibrosis, ROS and NO synthesis, glycolysis, and blood vessel morphogenesis. On the other hand, non-phagocytic macrophages enhance the expression of genes associated with migration, oxidative phosphorylation, and mitochondrial fission as well as anti-viral responses when compared to phagocytic macrophages. Notably, conversion from phagocytic to satiated macrophages is associated with a reduction in the expression of extracellular matrix constituents that were demonstrated to be associated with idiopathic pulmonary fibrosis (IPF). Thus, macrophage satiation during the resolution of inflammation seems to bring about a transcriptomic transition that resists tissue fibrosis and oxidative damage while promoting the restoration of tissue homeostasis to complete the resolution of inflammation.
Adenosine-to-inosine (A-to-I) RNA editing is an epigenetic modification catalyzed by adenosine deaminases acting on RNA (ADARs), and is especially prevalent in the brain. We used the highly accurate ...microfluidics-based multiplex PCR sequencing (mmPCR-seq) technique to assess the effects of development and environmental stress on A-to-I editing at 146 pre-selected, conserved sites in the rat prefrontal cortex and amygdala. Furthermore, we asked whether changes in editing can be observed in offspring of stress-exposed rats. In parallel, we assessed changes in ADARs expression levels.
In agreement with previous studies, we found editing to be generally higher in adult compared to neonatal rat brain. At birth, editing was generally lower in prefrontal cortex than in amygdala. Stress affected editing at the serotonin receptor 2c (Htr2c), and editing at this site was significantly altered in offspring of rats exposed to prereproductive stress across two generations. Stress-induced changes in Htr2c editing measured with mmPCR-seq were comparable to changes measured with Sanger and Illumina sequencing. Developmental and stress-induced changes in Adar and Adarb1 mRNA expression were observed but did not correlate with editing changes.
Our findings indicate that mmPCR-seq can accurately detect A-to-I RNA editing in rat brain samples, and confirm previous accounts of a developmental increase in RNA editing rates. Our findings also point to stress in adolescence as an environmental factor that alters RNA editing patterns several generations forward, joining a growing body of literature describing the transgenerational effects of stress.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Virtual collaborative Q&A communities generate shared knowledge through the interaction of people and content. This knowledge is often fragmented, and its value as a collective, collaboratively ...formed product, is largely overlooked. Inspired by work on individual mental semantic networks, the current study explores the networks formed by user-added associative links as reflecting an aspect of self-organization within the communities' collaborative knowledge sharing. Using eight Q&A topic-centered discussions from the Stack Exchange platform, it investigated how associative links form internal structures within the networks. Network analysis tools were used to derive topological indicator metrics of complex structures from associatively-linked networks. Similar metrics extracted from 1000 simulated randomly linked networks of comparable sizes and growth patterns were used to generate estimated sampling distributions through bootstrap resampling, and 99% confidence intervals were constructed for each metric. The discussion-network indicators were compared against these. Results showed that participant-added associative links largely led to networks that were more clustered, integrated, and included posts with more connections than those that would be expected in random networks of similar size and growth pattern. The differences were observed to increase over time. Also, the largest connected subgraphs within the discussion networks were found to be modular. Limited qualitative observations have also pointed to the impacts of external content-related events on the network structures. The findings strengthen the notion that the networks emerging from associative link sharing resemble other information networks that are characterized by internal structures suggesting self-organization, laying the ground for further exploration of collaborative linking as a form of collective knowledge organization. It underscores the importance of recognizing and leveraging this latent mechanism in both theory and practice.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, ...or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.
With the growing role of online multi-participant collaborations in shaping the academic, professional, and civic spheres, incorporating collaborative online practices in educational settings has ...become imperative. As more educators include such practices in their curricula, they are faced with new challenges. Assessment of collaborations, especially in larger groups, is particularly challenging. Assessing the quality of the collaborative “thought process” and its product is essential for both pedagogical and evaluative purposes. While traditional quantitative quality measures were designed for individual work or the aggregated work of individuals, capturing the complexity and the integrative nature of high-quality collaborative learning requires novel methodologies. Network analysis provides methods and tools that can identify, describe, and quantify non-linear and complex phenomena. This paper applies network analysis to the content created by students through large-scale online collaborative concept-mapping and explores how these can be applied for the assessment of the quality of a collective product. Quantitative network structure measures are introduced for this purpose. The application and the affordances of these metrics are demonstrated on data from six large-group online collaborative discussions from academic settings. The metrics presented here address the organization and the integration of the content and enable a comparison of collaborative discussions.
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