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1.
Proc Natl Acad Sci U S A ; 121(42): e2403953121, 2024 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-39388273

RESUMEN

Droplets of one fluid in a second, immiscible fluid are typically spherical in shape due to the interfacial tension between the two fluids. Shear forces can lead to droplet deformation when they are subjected to flow, and these effects can be further modified when the droplet is stabilized by a surfactant due to a flow-induced gradients in the surfactant concentration. An alternative method of stabilizing droplets is through the use of colloidal particles, whose stabilization behavior is intrinsically different from molecular surfactants. Under the same flow condition, a gradient of particle concentration can result in the jamming of particles in regions with a high packing density, making the interface solid-like, albeit only under compression and not tension. However, how this asymmetry in the surfactant properties alters the droplet shape under shear is unknown. Here, we show that shear of particle-stabilized droplets can lead to a remarkable array of shape deformations as the droplets flow through a constrained microchannel. The shear-induced migration of particles on the surface results in the formation of an elastic shell at the back of the droplet, which can wrinkle and invaginate, ultimately leading to a unique core-shell structure. The shapes depend on the Peclet number of the flow, reflecting the balance of shear forces that drive the particles and diffusion that randomizes them. These findings highlight the consequences of the asymmetry in the forces between the particles and provide a unique method to controllably create droplets with a vast array of different shapes.

2.
Phys Rev E ; 110(2-1): 024606, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39295063

RESUMEN

We introduce a technique, traction rheoscopy, to carry out mechanical testing of colloidal solids. A confocal microscope is used to directly measure stress and strain during externally applied deformation. The stress is measured, with single-mPa resolution, by determining the strain in a compliant polymer gel in mechanical contact with the colloidal solid. Simultaneously, the confocal microscope is used to measure structural change in the colloidal solid with single particle resolution during the deformation. To demonstrate the utility and sensitivity of this technique, we deform a hard-sphere colloidal glass in simple shear, and from the macroscopic shear strain and measured stress determine the stress-strain curve. Using the stress-strain curve and measured shear modulus, we decompose the macroscopic shear strain into an elastic and a plastic component. We also determine a local strain tensor for each particle using the changes in its nearest-neighbor distances. These local strains are spatially heterogeneous throughout the sample, but, when averaged, match the macroscopic strain. A microscopic yield criterion is used to split the local strains into subyield and yielded partitions; averages over these partitions complement the macroscopic elastic-plastic decomposition obtained from the stress-strain curve. By combining mechanical testing with single-particle structural measurements, traction rheoscopy is a unique tool for the study of deformation mechanisms in a diverse range of soft materials.

3.
Smart Med ; 3(2): e20240008, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-39188696

RESUMEN

Nucleic acid amplification testing (NAAT) remains one of the most reliable methods for pathogen identification. However, conventional bulk NAATs may not be sufficiently fast or sensitive enough for the detection of clinically-relevant pathogens in point-of-care testing. Here, we have developed a digital droplet RT-LAMP (ddRT-LAMP) assay that rapidly and quantitatively detects the SARS-CoV-2 viral E gene in microfluidic drops. Droplet partitioning using ddRT-LAMP significantly accelerates detection times across a wide range of template concentrations compared to bulk RT-LAMP assays. We discover that a reduction in droplet diameter decreases assay times up to a certain size, upon which surface adsorption of the RT-LAMP polymerase reduces reaction efficiency. Optimization of drop size and polymerase concentration enables rapid, sensitive, and quantitative detection of the SARS-CoV-2 E gene in only 8 min. These results highlight the potential of ddRT-LAMP assays as an excellent platform for quantitative point-of-care testing.

4.
Proc Natl Acad Sci U S A ; 121(31): e2404727121, 2024 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-39052829

RESUMEN

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. We present an experimental method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A unique gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. We demonstrate this method's efficacy by spiking two known viral genomes, Simian virus 40 (SV40, 5,243 bp) and Human Adenovirus 5 (HAd5, 35,938 bp), into a sewage sample with a final abundance in the droplets of around 0.1% and 0.015%, respectively. We achieve 100% recovery of the complete sequence of the spiked-in SV40 genome with uniform coverage distribution. For the larger HAd5 genome, we cover approximately 99.4% of its sequence. Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables single-genome whole-genome amplification and targeting characterizations of rare viral species and will facilitate our ability to access the mutational profile in single-virus genomes and contribute to an improved understanding of viral ecology.


Asunto(s)
Genoma Viral , Virus 40 de los Simios , Genoma Viral/genética , Virus 40 de los Simios/genética , Virus 40 de los Simios/aislamiento & purificación , Metagenómica/métodos , Humanos , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , Aguas del Alcantarillado/virología
5.
Soft Matter ; 20(23): 4633-4639, 2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38819788

RESUMEN

We formulate and characterize silicone gels near the gelation threshold with tunable refractive index, 1.4 < n < 1.49, and small viscoelastic moduli, G'∼1 Pa, for use in traction force microscopy. The near-critical gels have low-frequency storage plateau moduli between 50 Pa and 1 Pa, with loss moduli that are more than fifty times lower at low frequencies. The gels are linearly elastic up to strains of at least 50%. The refractive index of the gel is tuned to eliminate spherical aberrations during confocal imaging thereby minimizing signal loss when imaging through thick gel substrates. We also develop an index-matched colloidal particle, stabilized by a silicone brush, that can be dispersed throughout the gel. These particles can be used to determine the deformation of the gel. The combination of mechanical and optical properties of these near-critical gels extends the lower limit of stresses that can be measured with traction force microscopy to single mPa values, while minimizing optical aberrations.

6.
Nature ; 630(8017): 648-653, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38811735

RESUMEN

Colloidal crystals exhibit interesting properties1-4 that are in many ways analogous to their atomic counterparts. They have the same crystal structures2,5-7, undergo the same phase transitions8-10, and possess the same crystallographic defects11-14. In contrast to these structural properties, the mechanical properties of colloidal crystals are quite different from those of atomic systems. For example, unlike in atomic systems, the elasticity of hard-sphere colloidal crystals is purely entropic15; as a result, they are so soft that they can be melted just by stirring16,17. Moreover, crystalline materials deform plastically when subjected to increasing shear and become stronger because of the ubiquitous process of work hardening18; but this has so far never been observed in colloidal crystals, to our knowledge. Here we show that hard-sphere colloidal crystals exhibit work hardening. Moreover, despite their softness, the shear strength of colloidal crystals can increase and approach the theoretical limit for crystals, a value reached in very few other materials so far. We use confocal microscopy to show that the strength of colloidal crystals increases with dislocation density, and ultimately reaches the classic Taylor scaling behaviour for atomic materials19-21, although hard-sphere interactions lack the complexity of atomic interactions. We demonstrate that Taylor hardening arises through the formation of dislocation junctions22. The Taylor hardening regime, however, is established only after a transient phase, and it ceases when the colloidal crystals become so hard that the strain is localized within a thin boundary layer in which slip results from an unconventional motion of dislocations. The striking resemblance between colloidal and atomic crystals, despite the many orders of magnitude difference in particle size and shear modulus, demonstrates the universality of work hardening.


Asunto(s)
Coloides , Cristalización , Coloides/química , Microscopía Confocal , Resistencia al Corte , Dureza , Elasticidad
7.
Analyst ; 149(13): 3575-3584, 2024 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-38758107

RESUMEN

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route to expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient manner. We constructed a host bacterial cell to link the genotype of REs to the phenotype of ß-galactosidase expression based on the bacterial SOS response, and used a high-throughput microfluidic platform to isolate, detect and sort the REs in microfluidic drops at a frequency of ∼800 drops per second. We employed this strategy to screen for the XbaI gene from the constructed libraries of varied sizes. In a single round of sorting, a 90-fold target enrichment was achieved within 1 h. Compared to conventional RE-screening methods, the direct screening approach that we propose excels at efficient search of desirable REs in natural samples - especially unculturable samples - and can be tailored to high-throughput screening of a wide range of genotoxic targets.


Asunto(s)
Enzimas de Restricción del ADN , Escherichia coli , Respuesta SOS en Genética , Escherichia coli/genética , Escherichia coli/enzimología , Enzimas de Restricción del ADN/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/química , beta-Galactosidasa/metabolismo , beta-Galactosidasa/genética
8.
Proc Natl Acad Sci U S A ; 121(22): e2317227121, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38771870

RESUMEN

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.


Asunto(s)
Polisacáridos , Liposomas Unilamelares , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Polietilenglicoles/química , Colesterol/química , Colesterol/metabolismo , Lípidos/química
9.
Chemistry ; 30(43): e202400880, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38780896

RESUMEN

Directed evolution generates novel biomolecules with desired functions by iteratively diversifying the genetic sequence of wildtype biomolecules, relaying the genetic information to the molecule with function, and selecting the variants that progresses towards the properties of interest. While traditional directed evolution consumes significant labor and time for each step, continuous evolution seeks to automate all steps so directed evolution can proceed with minimum human intervention and dramatically shortened time. A major application of continuous evolution is the generation of novel enzymes, which catalyze reactions under conditions that are not favorable to their wildtype counterparts, or on altered substrates. The challenge to continuously evolve enzymes lies in automating sufficient, unbiased gene diversification, providing selection for a wide array of reaction types, and linking the genetic information to the phenotypic function. Over years of development, continuous evolution has accumulated versatile strategies to address these challenges, enabling its use as a general tool for enzyme engineering. As the capability of continuous evolution continues to expand, its impact will increase across various industries. In this review, we summarize the working mechanisms of recently developed continuous evolution strategies, discuss examples of their applications focusing on enzyme evolution, and point out their limitations and future directions.


Asunto(s)
Evolución Molecular Dirigida , Enzimas , Ingeniería de Proteínas , Enzimas/metabolismo , Enzimas/química , Enzimas/genética , Evolución Molecular Dirigida/métodos , Biocatálisis , Humanos
10.
Adv Mater ; 36(31): e2309860, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38615189

RESUMEN

Artificial antigen-presenting cells (aAPCs) are currently used to manufacture T cells for adoptive therapy in cancer treatment, but a readily tunable and modular system can enable both rapid T cell expansion and control over T cell phenotype. Here, it is shown that microgels with tailored surface biochemical properties can serve as aAPCs to mediate T cell activation and expansion. Surface functionalization of microgels is achieved via layer-by-layer coating using oppositely charged polymers, forming a thin but dense polymer layer on the surface. This facile and versatile approach is compatible with a variety of coating polymers and allows efficient and flexible surface-specific conjugation of defined peptides or proteins. The authors demonstrate that tethering appropriate stimulatory ligands on the microgel surface efficiently activates T cells for polyclonal and antigen-specific expansion. The expansion, phenotype, and functional outcome of primary mouse and human T cells can be regulated by modulating the concentration, ratio, and distribution of stimulatory ligands presented on microgel surfaces as well as the stiffness and viscoelasticity of the microgels.


Asunto(s)
Células Presentadoras de Antígenos , Microgeles , Propiedades de Superficie , Linfocitos T , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Humanos , Microgeles/química , Ratones , Proliferación Celular/efectos de los fármacos , Activación de Linfocitos , Polímeros/química
11.
Science ; 384(6692): 209-213, 2024 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-38603504

RESUMEN

Oil and water can only be mixed by dispersing droplets of one fluid in the other. When two droplets approach one another, the thin film that separates them invariably becomes unstable, causing the droplets to coalesce. The only known way to avoid this instability is through addition of a third component, typically a surfactant, which stabilizes the thin film at its equilibrium thickness. We report the observation that a thin fluid film of oil separating two water droplets can lead to an adhesive interaction between the droplets. Moreover, this interaction prevents their coalescence over timescales of several weeks, without the use of any surfactant or solvent.

12.
Proc Natl Acad Sci U S A ; 121(3): e2312031121, 2024 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-38194461

RESUMEN

The quantification and characterization of aggregated α-synuclein in clinical samples offer immense potential toward diagnosing, treating, and better understanding neurodegenerative synucleinopathies. Here, we developed digital seed amplification assays to detect single α-synuclein aggregates by partitioning the reaction into microcompartments. Using pre-formed α-synuclein fibrils as reaction seeds, we measured aggregate concentrations as low as 4 pg/mL. To improve our sensitivity, we captured aggregates on antibody-coated magnetic beads before running the amplification reaction. By first characterizing the pre-formed fibrils with transmission electron microscopy and size exclusion chromatography, we determined the specific aggregates targeted by each assay platform. Using brain tissue and cerebrospinal fluid samples collected from patients with Parkinson's Disease and multiple system atrophy, we demonstrated that the assay can detect endogenous pathological α-synuclein aggregates. Furthermore, as another application for these assays, we studied the inhibition of α-synuclein aggregation in the presence of small-molecule inhibitors and used a custom image analysis pipeline to quantify changes in aggregate growth and filament morphology.


Asunto(s)
Atrofia de Múltiples Sistemas , Enfermedad de Parkinson , Sinucleinopatías , Humanos , alfa-Sinucleína , Anticuerpos
13.
Lab Chip ; 24(5): 1135-1153, 2024 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-38165829

RESUMEN

Over the past two decades, advances in droplet-based microfluidics have facilitated new approaches to process and analyze samples with unprecedented levels of precision and throughput. A wide variety of applications has been inspired across multiple disciplines ranging from materials science to biology. Understanding the dynamics of droplets enables optimization of microfluidic operations and design of new techniques tailored to emerging demands. In this review, we discuss the underlying physics behind high-throughput generation and manipulation of droplets. We also summarize the applications in droplet-derived materials and droplet-based lab-on-a-chip biotechnology. In addition, we offer perspectives on future directions to realize wider use of droplet microfluidics in industrial production and biomedical analyses.

14.
Langmuir ; 40(3): 1950-1960, 2024 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-37991242

RESUMEN

Core-shell hydrogel microcapsules have sparked great interest due to their unique characteristics and prospective applications in the medical, pharmaceutical, and cosmetic fields. However, complex synthetic procedures and expensive costs have limited their practical application. Herein, we designed and prepared several multichannel and multijunctional droplet microfluidic devices based on soft lithography for the effective synthesis of core-shell hydrogel microcapsules for different purposes. Additionally, two different cross-linking processes (ultraviolet (UV) exposure and interfacial polymerization) were used to synthesize different types of core-shell structured hydrogel microcapsules. Hydrogel microcapsules with gelatin methacryloyl (GelMA) as the core and polyacrylamide (PAM) as the thin shell were synthesized using UV cross-linking. Using an interfacial polymerization process, another core-shell structured microcapsule with GelMA as the core and Ca2+ cross-linked alginate with polyethylenimine (PEI) as the shell was constructed, and the core diameter and total droplet diameter were flexibly controlled by carving. Noteworthy, these hydrogel microcapsules exhibit stimuli-responsiveness and controlled release ability. Overall, a novel technique was developed to successfully synthesize various hydrogel microcapsules with core-shell microstructures. The hydrogel microcapsules possess a multilayered structure that facilitates the coassembly of cells and drugs, as well as the layered assembly of multiple drugs, to develop synergistic therapeutic regimens. These adaptable and controllable hydrogel microdroplets shall held great promise for multicell or multidrug administration as well as for high-throughput drug screening.


Asunto(s)
Alginatos , Hidrogeles , Hidrogeles/química , Cápsulas/química , Alginatos/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química
15.
bioRxiv ; 2023 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-38014300

RESUMEN

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method's efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

16.
Proc Natl Acad Sci U S A ; 120(40): e2304272120, 2023 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-37774096

RESUMEN

Addition of particles to a viscoelastic suspension dramatically alters the properties of the mixture, particularly when it is sheared or otherwise processed. Shear-induced stretching of the polymers results in elastic stress that causes a substantial increase in measured viscosity with increasing shear, and an attractive interaction between particles, leading to their chaining. At even higher shear rates, the flow becomes unstable, even in the absence of particles. This instability makes it very difficult to determine the properties of a particle suspension. Here, we use a fully immersed parallel plate geometry to measure the high-shear-rate behavior of a suspension of particles in a viscoelastic fluid. We find an unexpected separation of the particles within the suspension resulting in the formation of a layer of particles in the center of the cell. Remarkably, monodisperse particles form a crystalline layer which dramatically alters the shear instability. By combining measurements of the velocity field and torque fluctuations, we show that this solid layer disrupts the flow instability and introduces a single-frequency component to the torque fluctuations that reflects a dominant velocity pattern in the flow. These results highlight the interplay between particles and a suspending viscoelastic fluid at very high shear rates.

17.
Nat Commun ; 14(1): 5760, 2023 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-37717044

RESUMEN

The severe difficulty to resolve simultaneously both the macroscopic deformation process and the dislocation dynamics on the atomic scale limits our understanding of crystal plasticity. Here we use colloidal crystals, imaged on the single particle level by high-speed three-dimensional (3D) confocal microscopy, and resolve in real-time both the relaxation of the epitaxial misfit strain and the accompanying evolution of dislocations. We show how dislocation interactions give rise to the formation of complex dislocation networks in 3D and to unexpectedly sharp plastic relaxation. The sharp relaxation is facilitated by attractive interactions that promote the formation of new dislocations that are more efficient in mediating strain. Dislocation networks form fragmented structures, as dislocation growth is blocked by either attractive interactions, which result in the formation of sessile dislocation junctions, or by repulsion from perpendicular segments. The strength of these blocking mechanisms decreases with the thickness of the crystal film. These results reveal the critical role of dislocation interactions in plastic deformation of thin films and can be readily generalized from the colloidal to the atomic scale.

18.
Proc Natl Acad Sci U S A ; 120(33): e2301366120, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37549257

RESUMEN

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.


Asunto(s)
Condensados Biomoleculares , Agregación Patológica de Proteínas , Humanos , Microscopía Confocal , Reología , Proteína FUS de Unión a ARN
19.
Nat Commun ; 14(1): 5130, 2023 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-37612289

RESUMEN

Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.


Asunto(s)
Escherichia coli , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Escherichia coli/genética , RNA-Seq , Antibacterianos/farmacología , Cartilla de ADN , ARN Ribosómico/genética
20.
Cell Syst ; 14(6): 464-481.e7, 2023 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-37348462

RESUMEN

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.


Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Transcriptoma/genética , Cinética , Organoides , Física
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