Hematopoietic stem cells (HSCs) give rise to diverse cell types in the blood system, yet our molecular understanding of the early trajectories that generate this enormous diversity in humans remains ...incomplete. Here, we leverage Drop‐seq, a massively parallel single‐cell RNA sequencing (scRNA‐seq) approach, to individually profile 20,000 progenitor cells from human cord blood, without prior enrichment or depletion for individual lineages based on surface markers. Our data reveal a transcriptional compendium of progenitor states in human cord blood, representing four committed lineages downstream from HSC, alongside the transcriptional dynamics underlying fate commitment. We identify intermediate stages that simultaneously co‐express “primed” programs for multiple downstream lineages, and also observe striking heterogeneity in the early molecular transitions between myeloid subsets. Integrating our data with a recently published scRNA‐seq dataset from human bone marrow, we illustrate the molecular similarity between these two commonly used systems and further explore the chromatin dynamics of “primed” transcriptional programs based on ATAC‐seq. Finally, we demonstrate that Drop‐seq data can be utilized to identify new heterogeneous surface markers of cell state that correlate with functional output.
Synopsis
Single‐cell transcriptome profiling of hematopoietic progenitors collected from human cord blood provides molecular evidence for multi‐lineage transcriptomic priming in early progenitors, which correlates with epigenetic state, surface marker expression, and functional output.
Unsupervised reconstruction of transcriptomic cell states in human CD34+ hematopoietic progenitors from cord blood is performed using Drop‐seq.
“Primed” and “de novo” programs that accompany specification into four downstream lineages are identified.
Integration of cord blood and bone marrow single cell datasets reveals strong conservation of molecular programs.
Heterogeneous surface markers are identified within early lymphoid‐primed multipotent progenitors (LMPPs) and their expression correlates with transcriptomic state and functional potential.
Single‐cell transcriptome profiling of hematopoietic progenitors collected from human cord blood provides molecular evidence for multi‐lineage transcriptomic priming in early progenitors, which correlates with epigenetic state, surface marker expression, and functional output.
Deep sedimentary basins amplify long‐period shaking from seismic waves, increasing the seismic hazard for cities sited on such basins. We perform 3‐D simulations of point source earthquakes ...distributed around the Seattle and Tacoma basins in Washington State to examine the dependence of basin amplification on source azimuth, depth, and earthquake type. For periods between 1 and 10 s, the pattern of amplification is spatially heterogeneous and differs considerably with the source‐to‐site azimuth. For close‐in earthquakes, the greatest basin amplification occurs toward the farside of the basin and ground motions from crustal earthquakes experience greater amplification than those from more vertically incident, deeper intraplate earthquakes. Love and Rayleigh waves form similar spatial patterns for a given source location, although the magnitude of amplification varies. The source dependence of basin amplification is an important factor for seismic hazard assessment, in both the Seattle and Tacoma basins, and by extension for deep sedimentary basins worldwide.
Plain Language Summary
Sedimentary basins amplify the ground shaking due to an earthquake. This increases the seismic hazard for cities located above such basins. Therefore, determining the amount and distribution of shaking amplification is critical to estimating the hazard associated with future earthquakes. We use computer simulations to determine how an earthquake's location, depth, and focal mechanism affect ground shaking for communities above the Seattle and Tacoma sedimentary basins, Washington State. We find that the amount of shaking amplification strongly depends on where the earthquake is located, as well as the earthquake type. This has important implications for seismic safety (e.g., for buildings and infrastructure) located above deep sedimentary basins worldwide.
Key Points
Basin amplification within the Seattle and Tacoma basins is strongly dependent on earthquake‐to‐site azimuth, source mechanism, and depth
For close‐in sources, peak long‐period basin amplification is generally focused on the farside of the basin relative to the source
Impact of source‐dependent basin amplification needs to be considered for estimates of seismic hazard in sedimentary basins worldwide
We describe the flexible multimethod seismic site characterization technique for obtaining shear-wave velocity (
V
S
) profiles and derivative information, such as the time-averaged
V
S
of the upper ...30 m (
V
S30
). Simply stated, the multimethod approach relies on the application of multiple independent noninvasive site characterization acquisition and analysis techniques utilized in a flexible field-based approach through a decision process based on primary factors such as the investigator’s available equipment, field logistics (e.g., available array deployment space, urban versus rural), geologic setting, and funding among other primary factors. In a multimethod approach, sites ideally should be characterized using both active and passive noninvasive (i.e., no drilling and only minimal disturbance to the ground surface) methods. Almost without exception, we recommend the use of at least one active-source technique for analyzing surface waves, which in the current state of practice would include one or more of the following: spectral analysis of surface waves (SASW; commonly Rayleigh waves) and multichannel analysis of surface waves (MASW; Rayleigh and/or Love waves). In addition, passive-source microtremor array methods (MAMs) using linear (one-dimensional) and two-dimensional arrays may be appropriate or even required for characterizing a given site. Similarly, the microtremor horizontal-to-vertical spectral ratio (mHVSR) method thacan be valuable for identifying shallow rock sites. Finally, P- and SH-wave seismic refraction methods warrant consideration, especially at rock and shallow-rock sites.
Droplet-based single-cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While this approach offers the exciting promise to deconvolute cellular heterogeneity ...in diseased tissues, the lack of cost-effective and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we developed a 3D-printed, low-cost droplet microfluidic control instrument and deploy it in a clinical environment to perform single-cell transcriptome profiling of disaggregated synovial tissue from five rheumatoid arthritis patients. We sequence 20,387 single cells revealing 13 transcriptomically distinct clusters. These encompass an unsupervised draft atlas of the autoimmune infiltrate that contribute to disease biology. Additionally, we identify previously uncharacterized fibroblast subpopulations and discern their spatial location within the synovium. We envision that this instrument will have broad utility in both research and clinical settings, enabling low-cost and routine application of microfluidic techniques.
Microtremor array measurements, and passive surface wave methods in general, have been increasingly used to non-invasively estimate shear-wave velocity structures for various purposes. The methods ...estimate dispersion curves and invert them for retrieving S-wave velocity profiles. This paper summarizes principles, limitations, data collection, and processing methods. It intends to enable students and practitioners to understand the principles needed to plan a microtremor array investigation, record and process the data, and evaluate the quality of investigation result. The paper focuses on the spatial autocorrelation processing method among microtremor array processing methods because of its relatively simple calculation and stable applicability.
Highlights
1. A summary of fundamental principles of calculating phase velocity from ambient noise
2. General recommendations for MAM data collection and processing using SPAC methods
3. A discussion of limitations and uncertainties in the methods
Droplet microfluidics has emerged as a promising technique to perform high-throughput, massively-parallel chemical and molecular biological reactions. Droplet microfluidic operations such as droplet ...generation, sorting, and fluid addition are well established; however, fluid exchange (i.e. washing) at high-throughput is challenging to implement. Here we present a microfluidic device architecture that utilizes wash buffer injection preceding a splitting junction in proximity to a magnetic field to transfer paramagnetic microparticles across a concentration gradient within a single droplet. The device can operate at high throughput (50 Hz) while preserving input droplet volume at the collection outlet as verified using high speed imaging. Using a two-stage device, combined microparticle retention rates (up to 97.5%) and high wash efficiency (92.9%) is demonstrated using dye absorbance and fluorescence. This method can be performed in a serial array to obtain an arbitrary degree of wash efficiency and integrated into lab-on-a-chip systems for use in multi-step microfluidic bioassays or single-cell genomic applications requiring high-fidelity washing steps within droplets.
Island Chain Stephenson, William
2019, 2019-07-18
eBook
The Cheshire Prize for Literature was inaugurated in 2003 as the High Sheriff's Cheshire Prize for Literature. It is funded by MBNA and is administered by the University of Chester. The 2018 ...competition was for short stories, and this collection contains stories by 21 of the shortlisted entries, including those by winners and runners up.
Unlocked Poole, Simon E; Stephenson, William
2021
eBook
Who could have anticipated the vicissitudes of the last year? And while the stark changes in our lives were pulling us together as a society, as we coped with what was unfolding, the quieter, often ...isolated time that followed allowed many to focus on writing. Lockdowns across the country may have created all kinds of problems for different people, but one of the positives that seems to have been unlocked across our county, and very probably across the country, was our individual creative potential. These pages are just one example of those isolated endeavours coming together into a collective expression of individual experience. This anthology is an incredibly unique publication, not only for how it documents this strange moment in time, but more importantly for how it reminds us of our need to explore, unravel, pose 'what-ifs', in order to make sense of the world: and the benefits of writing for our own wellbeing.
Accurate detection and quantification of mRNA isoforms from nanopore long-read sequencing remains challenged by technical noise, particularly in single cells. To address this, we introduce Isosceles, ...a computational toolkit that outperforms other methods in isoform detection sensitivity and quantification accuracy across single-cell, pseudo-bulk and bulk resolution levels, as demonstrated using synthetic and biologically-derived datasets. Here we show Isosceles improves the fidelity of single-cell transcriptome quantification at the isoform-level, and enables flexible downstream analysis. As a case study, we apply Isosceles, uncovering coordinated splicing within and between neuronal differentiation lineages. Isosceles is suitable to be applied in diverse biological systems, facilitating studies of cellular heterogeneity across biomedical research applications.Analysis of nanopore long-read sequencing is challenged by technical noise, particularly in single cells. Here, authors introduce Isosceles, a toolkit for accurate isoform detection, quantification, and flexible downstream analysis of long-read data at single-cell, pseudo-bulk and bulk resolutions.
Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In ...addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2′-O-methyl and base modifications across E. coli and S. cerevisiae ribosomal RNAs as shifts in current signal and dwell times distally through interactions with the helicase motor protein. We further use the 2′-hydroxyl reactive SHAPE reagent acetylimidazole to probe RNA structure at the single-molecule level with readout by direct nanopore sequencing.
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Nanopore sequencing detects endogenous RNA modifications at a single-molecule level2′-O-methyl (Nm) and pseudouridine (Ψ) alter nanopore translocation kineticsAcIm is a small-adduct-generating SHAPE-MaP reagentAcIm enables full-length single-molecule structural profiling on RNA
Stephenson et al. employ direct RNA nanopore sequencing to detect endogenous and exogenous modifications on single RNA molecules. The authors demonstrate detection of endogenous 2′-O-methylation (Nm) on native ribosomal RNAs, confirming known modification patterns. They describe the development of nanoSHAPE, a method that involves exogenously labeling RNA with a small-adduct-generating chemical probe that can reveal RNA structure using long-read sequencing.
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