High-throughput immune repertoire sequencing is promising to lead to new statistical diagnostic tools for medicine and biology. Successful implementations of these methods require a correct ...characterization, analysis, and interpretation of these data sets. We present IGoR (Inference and Generation Of Repertoires)-a comprehensive tool that takes B or T cell receptor sequence reads and quantitatively characterizes the statistics of receptor generation from both cDNA and gDNA. It probabilistically annotates sequences and its modular structure can be used to investigate models of increasing biological complexity for different organisms. For B cells, IGoR returns the hypermutation statistics, which we use to reveal co-localization of hypermutations along the sequence. We demonstrate that IGoR outperforms existing tools in accuracy and estimate the sample sizes needed for reliable repertoire characterization.
The early Drosophila embryo is an ideal model to understand the transcriptional regulation of well-defined patterns of gene expression in a developing organism 1. In this system, snapshots of ...transcription measurements obtained by RNA FISH on fixed samples 2, 3 cannot provide the temporal resolution needed to distinguish spatial heterogeneity 3 from inherent noise 4, 5. Here, we used the MS2-MCP system 6, 7 to visualize in living embryos nascent transcripts expressed from the canonical hunchback (hb) promoter 8, 9 under the control of Bicoid (Bcd) 10. The hb-MS2 reporter is expressed as synchronously as endogenous hb in the anterior half of the embryo, but unlike hb it is also active in the posterior, though more heterogeneously and more transiently than in the anterior. The length and intensity of active transcription periods in the anterior are strongly reduced in absence of Bcd, whereas posterior ones are mostly Bcd independent. This posterior noisy signal decreases progressively through nuclear divisions, so that the MS2 reporter expression mimics the known anterior hb pattern at cellularization. We propose that the establishment of the hb pattern relies on Bcd-dependent lengthening of transcriptional activity periods in the anterior and may require two distinct repression mechanisms in the posterior.
Display omitted
•Transcription at the hunchback promoter is visualized in early living embryos•The Bicoid transcription factor increases the duration of anterior activity periods•Short Bicoid-independent transcription periods are also detected posteriorly•An unexpected repression mechanism insures early formation of the expression border
Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously ...generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.
Display omitted
•Generation of hiPS cell-derived 3D spheroids resembling the hindbrain/spinal cord•Synaptically connected corticofugal projections in fused cortico-spinal assembloids•Stimulation of cortical neurons controls skeletal muscle in three-part assembloid•Cellular and functional changes in cortico-motor assembloids maintained long term
The assembly of 3D cultures derived from hiPS cells resembling cerebral cortex, hindbrain/spinal cord, and skeletal muscle form neural circuits that can be readily manipulated to model cortical control of muscle contraction in vitro long term.
Adaptive immunity's success relies on the extraordinary diversity of protein receptors on B and T cell membranes. Despite this diversity, the existence of public receptors shared by many individuals ...gives hope for developing population-wide vaccines and therapeutics. Using probabilistic modeling, we show many of these public receptors are shared by chance in healthy individuals. This predictable overlap is driven not only by biases in the random generation process of receptors, as previously reported, but also by their common functional selection. However, the model underestimates sharing between repertoires of individuals infected with SARS-CoV-2, suggesting strong specific antigen-driven convergent selection. We exploit this discrepancy to identify COVID-associated receptors, which we validate against datasets of receptors with known viral specificity. We study their properties in terms of sequence features and network organization, and use them to design an accurate diagnostic tool for predicting SARS-CoV-2 status from repertoire data.
Genesis of the αβ T-cell receptor Dupic, Thomas; Marcou, Quentin; Walczak, Aleksandra M ...
PLoS computational biology,
03/2019, Letnik:
15, Številka:
3
Journal Article
Recenzirano
Odprti dostop
The T-cell (TCR) repertoire relies on the diversity of receptors composed of two chains, called α and β, to recognize pathogens. Using results of high throughput sequencing and computational ...chain-pairing experiments of human TCR repertoires, we quantitively characterize the αβ generation process. We estimate the probabilities of a rescue recombination of the β chain on the second chromosome upon failure or success on the first chromosome. Unlike β chains, α chains recombine simultaneously on both chromosomes, resulting in correlated statistics of the two genes which we predict using a mechanistic model. We find that ∼35% of cells express both α chains. Altogether, our statistical analysis gives a complete quantitative mechanistic picture that results in the observed correlations in the generative process. We learn that the probability to generate any TCRαβ is lower than 10(-12) and estimate the generation diversity and sharing properties of the αβ TCR repertoire.
Marine sediments from the North Pacific document two episodes of expansion and strengthening of the subsurface oxygen minimum zone (OMZ) accompanied by seafloor hypoxia during the last deglacial ...transition. The mechanisms driving this hypoxia remain under debate. We present a new high-resolution alkenone palaeotemperature reconstruction from the Gulf of Alaska that reveals two abrupt warming events of 4-5 degrees Celsius at the onset of the Bølling and Holocene intervals that coincide with sudden shifts to hypoxia at intermediate depths. The presence of diatomaceous laminations and hypoxia-tolerant benthic foraminiferal species, peaks in redox-sensitive trace metals, and enhanced (15)N/(14)N ratio of organic matter, collectively suggest association with high export production. A decrease in (18)O/(16)O values of benthic foraminifera accompanying the most severe deoxygenation event indicates subsurface warming of up to about 2 degrees Celsius. We infer that abrupt warming triggered expansion of the North Pacific OMZ through reduced oxygen solubility and increased marine productivity via physiological effects; following initiation of hypoxia, remobilization of iron from hypoxic sediments could have provided a positive feedback on ocean deoxygenation through increased nutrient utilization and carbon export. Such a biogeochemical amplification process implies high sensitivity of OMZ expansion to warming.
Statistical mechanics for natural flocks of birds Bialek, William; Cavagna, Andrea; Giardina, Irene ...
Proceedings of the National Academy of Sciences - PNAS,
03/2012, Letnik:
109, Številka:
13
Journal Article
Recenzirano
Odprti dostop
Flocking is a typical example of emergent collective behavior, where interactions between individuals produce collective patterns on the large scale. Here we show how a quantitative microscopic ...theory for directional ordering in a flock can be derived directly from field data. We construct the minimally structured (maximum entropy) model consistent with experimental correlations in large flocks of starlings. The maximum entropy model shows that local, pairwise interactions between birds are sufficient to correctly predict the propagation of order throughout entire flocks of starlings, with no free parameters. We also find that the number of interacting neighbors is independent of flock density, confirming that interactions are ruled by topological rather than metric distance. Finally, by comparing flocks of different sizes, the model correctly accounts for the observed scale invariance of long-range correlations among the fluctuations in flight direction.
How a well-adapted immune system is organized Mayer, Andreas; Balasubramanian, Vijay; Mora, Thierry ...
Proceedings of the National Academy of Sciences - PNAS,
05/2015, Letnik:
112, Številka:
19
Journal Article
Recenzirano
Odprti dostop
The repertoire of lymphocyte receptors in the adaptive immune system protects organisms from diverse pathogens. A well-adapted repertoire should be tuned to the pathogenic environment to reduce the ...cost of infections. We develop a general framework for predicting the optimal repertoire that minimizes the cost of infections contracted from a given distribution of pathogens. The theory predicts that the immune system will have more receptors for rare antigens than expected from the frequency of encounters; individuals exposed to the same infections will have sparse repertoires that are largely different, but nevertheless exploit cross-reactivity to provide the same coverage of antigens; and the optimal repertoires can be reached via the dynamics of competitive binding of antigens by receptors and selective amplification of stimulated receptors. Our results follow from a tension between the statistics of pathogen detection, which favor a broader receptor distribution, and the effects of cross-reactivity, which tend to concentrate the optimal repertoire onto a few highly abundant clones. Our predictions can be tested in high-throughput surveys of receptor and pathogen diversity.
Significance The adaptive immune system uses the experience of past infections to prepare its limited repertoire of specialized receptors to protect organisms from future threats. What is the best way of doing this? Building a theoretical framework from first principles, we predict the composition of receptor repertoires that are optimally adapted to minimize the cost of infections from a given pathogenic environment. A naive repertoire can reach these optima through a biologically plausible competitive mechanism. Our findings explain how limited populations of immune receptors can self-organize to provide effective immunity against highly diverse pathogens. Our results also inform the design and interpretation of experiments surveying immune repertoires.
Hypervariable T cell receptors (TCRs) play a key role in adaptive immunity, recognizing a vast diversity of pathogen-derived antigens. Our ability to extract clinically relevant information from ...large high-throughput sequencing of TCR repertoires (RepSeq) data is limited, because little is known about TCR-disease associations. We present Antigen-specific Lymphocyte Identification by Clustering of Expanded sequences (ALICE), a statistical approach that identifies TCR sequences actively involved in current immune responses from a single RepSeq sample and apply it to repertoires of patients with a variety of disorders - patients with autoimmune disease (ankylosing spondylitis AS), under cancer immunotherapy, or subject to an acute infection (live yellow fever YF vaccine). We validate the method with independent assays. ALICE requires no longitudinal data collection nor large cohorts, and it is directly applicable to most RepSeq datasets. Its results facilitate the identification of TCR variants associated with diseases and conditions, which can be used for diagnostics and rational vaccine design.
The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated ...multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.
PDF
