RESUMEN
Proximity sequencing (Prox-seq) simultaneously measures gene expression, protein expression and protein complexes on single cells. Using information from dual-antibody binding events, Prox-seq infers surface protein dimers at the single-cell level. Prox-seq provides multi-dimensional phenotyping of single cells in high throughput, and was recently used to track the formation of receptor complexes during cell signaling and discovered a novel interaction between CD9 and CD8 in naïve T cells. The distribution of protein abundance can affect identification of protein complexes in a complicated manner in dual-binding assays like Prox-seq. These effects are difficult to explore with experiments, yet important for accurate quantification of protein complexes. Here, we introduce a physical model of Prox-seq and computationally evaluate several different methods for reducing background noise when quantifying protein complexes. Furthermore, we developed an improved method for analysis of Prox-seq data, which resulted in more accurate and robust quantification of protein complexes. Finally, our Prox-seq model offers a simple way to investigate the behavior of Prox-seq data under various biological conditions and guide users toward selecting the best analysis method for their data.
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Comunicación Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.
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ADN Viral , Genoma Viral , Herpesvirus Humano 1 , Mutación , ADN Viral/genética , Genoma Viral/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crecimiento & desarrollo , Replicación Viral , Células Eucariotas/virología , Núcleo Celular/virología , Pase Seriado , HumanosRESUMEN
We present a simple method for on-demand manipulation of aqueous droplets in oil. With numerical simulations and experiments, we show that a vibrating membrane can produce acoustic streaming. By making use of this vortical flow, we manage to repulse the droplets away from the membrane edges. Then, by simply aligning the membrane at 45° to the flow, the droplets can be forced to follow the membrane's boundaries, thus steering them across streamlines and even between different oil types. We also characterize the repulsion and steering effect with various excitation voltages at different water and oil flow rates. The maximum steering frequency we have achieved is 165 Hz. The system is extremely robust and reliable: the same membrane can be reused after many days and with different oils and/or surfactants at the same operating frequency.
RESUMEN
Complex cellular functions occur via the coordinated formation and dissociation of protein complexes. Functions such as the response to a signaling ligand can incorporate dozens of proteins and hundreds of complexes. Until recently, it has been difficult to measure multiple protein complexes at the single-cell level. Here, we present a step-by-step procedure for proximity sequencing, which enables the simultaneous measurement of proteins, mRNA and hundreds of protein complexes located on the outer membrane of cells. We guide the user through probe creation, sample preparation, staining, sequencing and computational quantification of protein complexes. This protocol empowers researchers to study, for example, the interplay between transcriptional states and cellular functions by coupling measurements of transcription to measurements of linked effector molecules, yet could be generalizable to other paired events. The protocol requires roughly 16 h spread over several days to complete by users with expertise in basic molecular biology and single-cell sequencing.
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Age is a major risk factor for severe coronavirus disease 2019 (COVID-19), yet the mechanisms behind this relationship have remained incompletely understood. To address this, we evaluated the impact of aging on host immune response in the blood and the upper airway, as well as the nasal microbiome in a prospective, multicenter cohort of 1031 vaccine-naïve patients hospitalized for COVID-19 between 18 and 96 years old. We performed mass cytometry, serum protein profiling, anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays, and blood and nasal transcriptomics. We found that older age correlated with increased SARS-CoV-2 viral abundance upon hospital admission, delayed viral clearance, and increased type I interferon gene expression in both the blood and upper airway. We also observed age-dependent up-regulation of innate immune signaling pathways and down-regulation of adaptive immune signaling pathways. Older adults had lower naïve T and B cell populations and higher monocyte populations. Over time, older adults demonstrated a sustained induction of pro-inflammatory genes and serum chemokines compared with younger individuals, suggesting an age-dependent impairment in inflammation resolution. Transcriptional and protein biomarkers of disease severity differed with age, with the oldest adults exhibiting greater expression of pro-inflammatory genes and proteins in severe disease. Together, our study finds that aging is associated with impaired viral clearance, dysregulated immune signaling, and persistent and potentially pathologic activation of pro-inflammatory genes and proteins.
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COVID-19 , Humanos , Anciano , Adolescente , Adulto Joven , Adulto , Persona de Mediana Edad , Anciano de 80 o más Años , SARS-CoV-2 , Estudios Prospectivos , Multiómica , QuimiocinasRESUMEN
Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples.
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Formaldehído/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Polímeros/química , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Células 3T3 , Células A549 , Animales , Línea Celular Tumoral , Citometría de Flujo/métodos , Células HEK293 , Humanos , Ratones , ARN/análisis , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
This study presents a novel acoustic mixer comprising of a microfabricated silicon nitride membrane with a hole etched through it. We show that the introduction of the through hole leads to extremely fast and homogeneous mixing. When the membrane is immersed in fluid and subjected to acoustic excitation, a strong streaming field in the form of vortices is generated. The vortices are always observed to centre at the hole, pointing to the critical role it has on the streaming field. We hypothesise that the hole introduces a discontinuity to the boundary conditions of the membrane, leading to strong streaming vortices. With numerical simulations, we show that the hole's presence can increase the volume force responsible for driving the streaming field by 2 orders of magnitude, thus supporting our hypothesis. We investigate the mixing performance at different Peclet numbers by varying the flow rates for various devices containing circular, square and rectangular shaped holes of different dimensions. We demonstrate rapid mixing within 3 ms mixing time (90% mixing efficiency at 60 µl min(-1) total flow rate, Peclet number equals 8333 ± 3.5%) is possible with the current designs. Finally, we examine the membrane with two circular holes which are covered by air bubbles and compare it to when the membrane is fully immersed. We find that coupling between the holes' vortices occurs only when membrane is immersed; while with the bubble membrane, the upstream hole's vortices can act as a blockage to fluid flow passing it.