Single-cell proteomics enabled by next-generation sequencing or mass spectrometry.

In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying heterogeneity of complex biological systems. Technological advances have also enabled protein measurements, further contributing to the elucidation of cell types and states present in complex tissues. Recently, there have been independent advances in mass spectrometric techniques bringing us one step closer to characterizing single-cell proteomes. Here we discuss the challenges of detecting proteins in single cells by both mass spectrometry and sequencing-based methods. We review the state of the art for these techniques and propose that there is a space for technological advancements and complementary approaches that maximize the advantages of both classes of technologies.

Rigidity percolation in a random tensegrity via analytic graph theory.

Functional structures from across the engineered and biological world combine rigid elements such as bones and columns with flexible ones such as cables, fibers, and membranes. These structures are known loosely as tensegrities, since these cable-like elements have the highly nonlinear property of supporting only extensile tension. Marginally rigid systems are of particular interest because the number of structural constraints permits both flexible deformation and the support of external loads. We present a model system in which tensegrity elements are added at random to a regular backbone. This system can be solved analytically via a directed graph theory, revealing a mechanical critical point generalizing that of Maxwell. We show that even the addition of a few cable-like elements fundamentally modifies the nature of this transition point, as well as the later transition to a fully rigid structure. Moreover, the tensegrity network displays a collective avalanche behavior, in which the addition of a single cable leads to the elimination of multiple floppy modes, a phenomenon that becomes dominant at the transition point. These phenomena have implications for systems with nonlinear mechanical constraints, from biopolymer networks to soft robots to jammed packings to origami sheets.

Transcriptional Dysregulation and Impaired Neuronal Activity in FMR1 Knock-Out and Fragile X Patients' iPSC-Derived Models.

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.

Simultaneous epitope and transcriptome measurement in single cells.

High-throughput single-cell RNA sequencing has transformed our understanding of complex cell populations, but it does not provide phenotypic information such as cell-surface protein levels. Here, we describe cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), a method in which oligonucleotide-labeled antibodies are used to integrate cellular protein and transcriptome measurements into an efficient, single-cell readout. CITE-seq is compatible with existing single-cell sequencing approaches and scales readily with throughput increases.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

Molecular transitions in early progenitors during human cord blood hematopoiesis.

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.

Combining temperature and force to study folding of an RNA hairpin.

RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG(0pN,T), the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG(0pN,T) and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG(0pN,T) depended linearly on temperature, yielding ΔH(exp) and ΔS(exp) that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.

The essential role of stacking adenines in a two-base-pair RNA kissing complex.

In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5'-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.

Diagnosis and ablation of multiform fascicular tachycardia.

INTRODUCTION: Fascicular tachycardia (FT) is an uncommon cause of monomorphic sustained ventricular tachycardia (VT). We describe 6 cases of FT with multiform QRS morphologies. METHODS AND RESULTS: Six of 823 consecutive VT cases were retrospectively analyzed and found attributable to FT with multiform QRS patterns, with 3 cases exhibiting narrow QRS VT as well. All underwent electrophysiology study including fascicular potential mapping, entrainment pacing, and electroanatomic mapping. The first 3 cases describe similar multiform VT patterns with successful ablation in the upper mid septum. Initially, a right bundle branch block (RBBB) VT with superior axis was induced. Radiofrequency catheter ablation (RFCA) targeting the left posterior fascicle (LPF) resulted in a second VT with RBBB inferior axis. RFCA in the upper septum just apical to the LBB potential abolished VT in all cases. Cases 4 and 5 showed RBBB VT with alternating fascicular block compatible with upper septal dependent VT, resulting in bundle branch reentrant VT (BBRT) after ablation of LPF and left anterior fascicle (LAF). Finally, Cases 5 and 6 demonstrated spontaneous shift in QRS morphology during VT, implicating participation of a third fascicle. In Case 6, successful ablation was achieved over the proximal LAF, likely representing insertion of the auxiliary fascicle near the proximal LAF. CONCLUSIONS: Multiform FTs show a reentrant mechanism using multiple fascicular branches. We hypothesize that retrograde conduction over the septal fascicle produces alternate fascicular patterns as well as narrow VT forms. Ablation of the respective fascicle was successful in abolishing FT but does not preclude development of BBRT unless septal fascicle is targeted and ablated.

The physical foundation of vasoocclusion in sickle cell disease.

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.

An open source toolkit for repurposing Illumina sequencing systems as versatile fluidics and imaging platforms.

Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument, comprising an epifluorescence microscope with integrated fluidics, into an open platform for programmable applications without need for specialized engineering or software development expertise. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. Given the widespread availability of HiSeq 2500 platforms and the simplicity of the modifications required to repurpose these systems, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.

Direct detection of RNA modifications and structure using single-molecule nanopore sequencing.

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.

Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low-cost microfluidic instrumentation.

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.

Unusual presentation of spinal lipomatosis.

Spinal epidural lipomatosis (SEL) is a rare condition characterized by overgrowth of normal adipose tissue in the extradural space within the spinal canal that can lead to significant spinal cord compression. It is most commonly reported in patients receiving chronic glucocorticoid therapy. Other causes can include obesity and hypercortisolism. Occasionally, idiopathic SEL will occur in patients with no known risk factors, but cases are more generally reported in obesity and males. We present the case of a 35 year-old non-obese woman found to have rapidly progressive SEL that was not associated with any of the common causes of the disorder.

Nanomanipulation of single RNA molecules by optical tweezers.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Southwest Oncology Group phase II study of arsenic trioxide in patients with refractory germ cell malignancies.

BACKGROUND: To the authors' knowledge, no satisfactory therapy is available for patients with refractory germ cell neoplasms. The activity and safety of arsenic trioxide in refractory germ cell tumors was assessed. METHODS: Twenty patients were treated with arsenic trioxide at a dose of 0.25 mg/kg/day and administered intravenously over 1 to 2 hours on Days 1-5 and repeated every 28 days. RESULTS: There were no complete or partial responses. The median progression-free survival was 1 month and the median overall survival was 2 months. Three patients died as a result of adverse events believed to be possibly related to treatment: ventricular arrhythmia, adult respiratory distress syndrome, and pneumonitis. CONCLUSIONS: In the current study, arsenic trioxide in the dose regimen and schedule employed was found to have no activity in men with refractory germ cell malignancies. Treatment was associated with severe toxicity. Dismal overall survival reflects the poor outcome in this patient group and highlights the acute need for new agents with activity in refractory germ cell neoplasms.

Waterborne Disease Outbreaks, 1986-1988.

From 1986 to 1988, 24 states and Puerto Rico reported 50 outbreaks of illness due to water that people intended to drink, affecting 25,846 persons. The protozoal parasite Giardia lamblia was the agent most commonly implicated in outbreaks, as it has been for the last 10 years; many of these outbreaks were associated with ingestion of chlorinated but unfiltered surface water. Shigella sonnei was the most commonly implicated bacterial pathogen; in outbreaks caused by this pathogen, water supplies were found to be contaminated with human waste. Cryptosporidium contamination of a chlorinated, filtered public water supply caused the largest outbreak during this period, affecting an estimated 13,000 persons. A large multistate outbreak caused by commercially produced ice made from contaminated well water caused illness with Norwalk-like virus among an estimated 5,000 persons. The first reported outbreak of chronic diarrhea of unknown cause associated with drinking untreated well water occurred in 1987. Twenty-six outbreaks due to recreational water use were also reported, including outbreaks of Pseudomonas dermatitis , associated with the use of hot tubs or whirlpools, and swimming-associated shigellosis, giardiasis, and viral illness. Although the total number of reported water-related outbreaks has been declining in recent years, the few large outbreaks due to Cryptosporidium , Norwalk-like agent, S. sonnei , and G. lamblia caused more cases of illness in 1987 than have been reported to the Water-Related Disease Outbreak Surveillance System for any other year since CDC and the Environmental Protection Agency began tabulating these data in 1971.