Adaptation of CUT&RUN for use in African trypanosomes.

This Cleavage Under Targets and Release Using Nuclease (CUT&RUN) protocol produces genomic occupancy data for a protein of interest in the protozoan parasite Trypanosoma brucei. The data produced is analyzed in a similar way as that produced by ChIP-seq. While we describe the protocol for parasites carrying an epitope tag for the protein of interest, antibodies against the native protein could be used for the same purpose.

A spatially resolved timeline of the human maternal-fetal interface.

Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR)1-3. However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.

Genomic Occupancy of the Bromodomain Protein Bdf3 Is Dynamic during Differentiation of African Trypanosomes from Bloodstream to Procyclic Forms.

Trypanosoma brucei, the causative agent of human and animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism during adaptation to each host environment. These changes are reflected in the different transcriptomes of parasites living in each host. However, it remains unclear whether chromatin-interacting proteins help mediate these changes. Bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stages remains unknown. To address this question, we performed cleavage under target and release using nuclease (CUT&RUN) against bromodomain protein 3 (Bdf3) in parasites differentiating from bloodstream to insect forms. We found that Bdf3 occupancy at most loci increased at 3 h following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes with altered transcript levels during differentiation, such as procyclins, procyclin-associated genes, and invariant surface glycoproteins. Most Bdf3-occupied sites are observed throughout differentiation. However, one site appears de novo during differentiation and lies proximal to the procyclin gene locus housing genes essential for remodeling surface proteins following transition to the insect stage. These studies indicate that occupancy of chromatin-interacting proteins is dynamic during life cycle stage transitions and provide the groundwork for future studies on the effects of changes in bromodomain protein occupancy. Additionally, the adaptation of CUT&RUN for Trypanosoma brucei provides other researchers with an alternative to chromatin immunoprecipitation (ChIP). IMPORTANCE The parasite Trypanosoma brucei is the causative agent of human and animal African trypanosomiasis (sleeping sickness). Trypanosomiasis, which affects humans and cattle, is fatal if untreated. Existing drugs have significant side effects. Thus, these parasites impose a significant human and economic burden in sub-Saharan Africa, where trypanosomiasis is endemic. T. brucei cycles between the mammalian host and a tsetse fly vector, and parasites undergo huge changes in morphology and metabolism to adapt to different hosts. Here, we show that DNA-interacting bromodomain protein 3 (Bdf3) shows changes in occupancy at its binding sites as parasites transition from the bloodstream to the insect stage. Additionally, a new binding site appears near the locus responsible for remodeling of parasite surface proteins during transition to the insect stage. Understanding the mechanisms behind host adaptation is important for understanding the life cycle of the parasite.

Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning.

A principal challenge in the analysis of tissue imaging data is cell segmentation-the task of identifying the precise boundary of every cell in an image. To address this problem we constructed TissueNet, a dataset for training segmentation models that contains more than 1 million manually labeled cells, an order of magnitude more than all previously published segmentation training datasets. We used TissueNet to train Mesmer, a deep-learning-enabled segmentation algorithm. We demonstrated that Mesmer is more accurate than previous methods, generalizes to the full diversity of tissue types and imaging platforms in TissueNet, and achieves human-level performance. Mesmer enabled the automated extraction of key cellular features, such as subcellular localization of protein signal, which was challenging with previous approaches. We then adapted Mesmer to harness cell lineage information in highly multiplexed datasets and used this enhanced version to quantify cell morphology changes during human gestation. All code, data and models are released as a community resource.