Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively ...parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.
Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed ...linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%–87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.
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•Linked-read genome sequencing of mCRPC resolves haplotypes and rearrangements•CDK12 inactivation is associated with a global tandem duplication phenotype•A majority of cases have duplications of an enhancer of the androgen receptor•Progression on androgen pathway inhibitors is associated with gains in AR and AR enhancer
Linked-read genome sequencing data from patients highlight that amplification of an enhancer upstream of the androgen receptor locus is a key feature of metastatic castration-resistant prostate cancer.
Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here, we sought to determine whether monocyte-derived ...macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH), we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next, we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance, worsened neurological recovery, exacerbated iron deposition, and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset, suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus, our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH.
Food allergy affects an estimated 8% of children in the United States. Oral immunotherapy (OIT) is a recently approved treatment, with outcomes ranging from sustained tolerance to food allergens to ...no apparent benefit. The immunological underpinnings that influence clinical outcomes of OIT remain largely unresolved. Using single-cell RNA-Seq and paired T cell receptor α/β (TCRα/β) sequencing, we assessed the transcriptomes of CD154+ and CD137+ peanut-reactive T helper (Th) cells from 12 patients with peanut allergy longitudinally throughout OIT. We observed expanded populations of cells expressing Th1, Th2, and Th17 signatures that further separated into 6 clonally distinct subsets. Four of these subsets demonstrated a convergence of TCR sequences, suggesting antigen-driven T cell fates. Over the course of OIT, we observed suppression of Th2 and Th1 gene signatures in effector clonotypes but not T follicular helper-like (Tfh-like) clonotypes. Positive outcomes were associated with stronger suppression of Th2 signatures in Th2A-like cells, while treatment failure was associated with the expression of baseline inflammatory gene signatures that were present in Th1 and Th17 cell populations and unmodulated by OIT. These results demonstrate that differential clinical responses to OIT are associated with both preexisting characteristics of peanut-reactive CD4+ T cells and suppression of a subset of Th2 cells.
Morphological control over the bulk heterojunction (BHJ) microstructure of a high‐efficiency small molecule photovoltaic system is demonstrated using both thermal treatment and solvent additive ...processing. Single crystal X‐ray diffraction is utilized to understand molecular interactions in the solid state and the BHJ morphology is examined using bright field, high‐resolution, and cross‐section transmission electron microscopy techniques. Controlling the domain size, while maintaining good molecular order within the semiconducting donor material, is found to be crucial in achieving high performance and over 90% internal quantum efficiency exhibited under the optimized conditions.
Morphological control over the bulk heterojunction (BHJ) microstructure of a small‐molecule photovoltaic system is demonstrated using thermal treatment and solvent additives. Single crystal X‐ray diffraction and transmission electron microscopy are utilized to investigate solid‐state interactions and the BHJ morphology. Domain size and molecular order are crucial to achieving the 7.0% power conversion efficiency and over 90% internal quantum efficiency exhibited under optimized conditions.
The complex heterogeneity of cells, and their interconnectedness with each other, are major challenges to identifying clinically relevant measurements that reflect the state and capability of the ...immune system. Highly multiplexed, single-cell technologies may be critical for identifying correlates of disease or immunological interventions as well as for elucidating the underlying mechanisms of immunity. Here we review limitations of bulk measurements and explore advances in single-cell technologies that overcome these problems by expanding the depth and breadth of functional and phenotypic analysis in space and time. The geometric increases in complexity of data make formidable hurdles for exploring, analyzing and presenting results. We summarize recent approaches to making such computations tractable and discuss challenges for integrating heterogeneous data obtained using these single-cell technologies.
High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. ...Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 (“Second-Strand Synthesis”), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.
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•Seq-Well S3 uses second-strand synthesis to improve transcript capture•Seq-Well S3 was benchmarked against a best-in-class commercial platform•Seq-Well S3 was applied to profile inflammatory cell states in skin diseases•Analysis of skin inflammation uncovered unique and conserved cellular phenotypes
Hughes et al. report the development of a technique for high-throughput single-cell RNA-sequencing, “Seq-Well S3,” that enables increased sensitivity and improved detection of genes including transcription factors, cytokines, and cytokine receptors. Using Seq-Well S3, the authors define inflammatory cell states across multiple skin diseases.
A long standing question in organic electronics concerns the effects of molecular orientation at donor/acceptor heterojunctions. Given a well-controlled donor/acceptor bilayer system, we uncover the ...genuine effects of molecular orientation on charge generation and recombination. These effects are studied through the point of view of photovoltaics-however, the results have important implications on the operation of all optoelectronic devices with donor/acceptor interfaces, such as light emitting diodes and photodetectors. Our findings can be summarized by two points. First, devices with donor molecules face-on to the acceptor interface have a higher charge transfer state energy and less non-radiative recombination, resulting in larger open-circuit voltages and higher radiative efficiencies. Second, devices with donor molecules edge-on to the acceptor interface are more efficient at charge generation, attributed to smaller electronic coupling between the charge transfer states and the ground state, and lower activation energy for charge generation.Molecular orientation profoundly affects the performance of donor-acceptor heterojunctions, whilst it has remained challenging to investigate the detail. Using a controllable interface, Ran et al. show that the edge-on geometries improve charge generation at the cost of non-radiative recombination loss.
High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ...ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4
T cell (T
2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.
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