Super-resolution imaging of linearized chromatin in tunable nanochannels.

Nanofluidic linearization and optical mapping of naked DNA have been reported in the research literature, and implemented in commercial instruments. However, the resolution with which DNA features can be resolved is still inherently limited by both Brownian motion and diffraction-limited optics. Direct analysis of native chromatin is further hampered by difficulty in electrophoretic manipulation, which is routinely used for DNA analysis. This paper describes the development of a three-layer, tunable, nanochannel system that enables non-electrophoretic linearization and immobilization of native chromatin. Furthermore, through careful selection of self-blinking fluorescent dyes and the design of the nanochannel system, we achieve direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging of the linearized chromatin. As an initial demonstration, rDNA chromatin extracted from Tetrahymena is analyzed by multi-color imaging of total DNA, newly synthesized DNA, and newly synthesized histone H3. Our analysis reveals a relatively even distribution of newly synthesized H3 across two halves of the rDNA chromatin with palindromic symmetry, supporting dispersive nucleosome segregation. As a proof-of-concept study, our work achieves super-resolution imaging of native chromatin fibers linearized and immobilized in tunable nanochannels. It opens up a new avenue for collecting long-range and high-resolution epigenetic information as well as genetic information.

Synthetic neutrophil extracellular traps dissect bactericidal contribution of NETs under regulation of α-1-antitrypsin.

Deciphering the complex interplay of neutrophil extracellular traps (NETs) with the surrounding environment is a challenge with notable clinical implications. To bridge the gap in knowledge, we report our findings on the antibacterial activity against Pseudomonas aeruginosa of synthetic NET-mimetic materials composed of nanofibrillated DNA-protein complexes. Our synthetic system makes component-by-component bottom-up analysis of NET protein effects possible. When the antimicrobial enzyme neutrophil elastase (NE) is incorporated into the bactericidal DNA-histone complexes, the resulting synthetic NET-like structure exhibits an unexpected reduction in antimicrobial activity. This critical immune function is rescued upon treatment with alpha-1-antitrypsin (AAT), a physiological tissue-protective protease inhibitor. This suggests a direct causal link between AAT inhibition of NE and preservation of histone-mediated antimicrobial activity. These results help better understand the complex and, at times, contradictory observations of in vivo antimicrobial effects of NETs and AAT by excluding neutrophil, cytokine, and chemoattractant contributions.

Nucleic acid partitioning in PEG-Ficoll protocells.

The phase separation of aqueous polymer solutions is a widely used method for producing self-assembled, membraneless droplet protocells. Non-ionic synthetic polymers forming an aqueous two-phase system (ATPS) have been shown to reliably form protocells that, when equipped with biological materials, are useful for applications such as analyte detection. Previous characterization of an ATPS-templated protocell did not investigate the effects of its biological components on phase stability. Here we report the phase diagram of a PEG 35k-Ficoll 400k-water ATPS at baseline and in the presence of necessary protocell components. Because the stability of an ATPS can be sensitive to small changes in composition, which in turn impacts solute partitioning, we present partitioning data of a variety of nucleic acids in response to protocell additives. The results show that the additives-particularly a mixture of salts and small organic molecules-have profound positive effects on ATPS stability and nucleic acid partitioning, both of which significantly contribute to protocell function. Our data uncovers several new areas of optimization for future protocell engineering.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Dosage-dependent antimicrobial activity of DNA-histone microwebs against Staphylococcus aureus.

Neutrophil extracellular traps (NETs) is an antimicrobial cobweb-structured material produced by immune cells for clearance of pathogens in the body, but paradoxically associated with biofilm formation and exacerbated lung infections. To provide a better materials perspective on the pleiotropic roles played by NETs at diverse compositions/concentrations, a NETs-like material (called 'microwebs', abbreviated as µwebs) is synthesized for decoding the antimicrobial activity of NETs against Staphylococcus aureus in infection-relevant conditions. We show that µwebs composed of low-to-intermediate concentrations of DNA-histone complexes successfully trap and inhibit S. aureus growth and biofilm formation. However, with growing concentrations and histone proportions, the resulting microwebs appear gel-like structures accompanied by reduced antimicrobial activity that can even promote formation of S. aureus biofilms. Our simplified model of NETs provides a materials-based evidence on NETs-relevant pathology in the development of biofilms.

Aqueous Two-Phase Systems and Microfluidics for Microscale Assays and Analytical Measurements.

Phase separation is a common occurrence in nature. Synthetic and natural polymers, salts, ionic liquids, surfactants, and biomacromolecules phase separate in water, resulting in an aqueous two-phase system (ATPS). This review discusses the properties, handling, and uses of ATPSs. These systems have been used for protein, nucleic acid, virus, and cell purification and have in recent years found new uses for small organics, polysaccharides, extracellular vesicles, and biopharmaceuticals. Analytical biochemistry applications such as quantifying protein-protein binding, probing for conformational changes, or monitoring enzyme activity have been performed with ATPSs. Not only are ATPSs biocompatible, they also retain their properties at the microscale, enabling miniaturization experiments such as droplet microfluidics, bacterial quorum sensing, multiplexed and point-of-care immunoassays, and cell patterning. ATPSs include coacervates and may find wider interest in the context of intracellular phase separation and origin of life. Recent advances in fundamental understanding and in commercial application are also considered.

Engineering cell heterogeneity into organs-on-a-chip.

Organ-on-a-chip development is an application that will benefit from advances in cell heterogeneity characterization because these culture models are intended to mimic in vivo microenvironments, which are complex and dynamic. Due in no small part to advances in microfluidic single cell analysis methods, cell-to-cell variability is an increasingly understood feature of physiological tissues, with cell types from as common as 1 out of every 2 cells to as rare as 1 out of every 100 000 cells having important roles in the biochemical and biological makeup of tissues and organs. Variability between neighboring cells can be transient or maintained, and ordered or stochastic. This review covers three areas of well-studied cell heterogeneity that are informative for organ-on-a-chip development efforts: tumors, the lung, and the intestine. Then we look at how recent single cell analysis strategies have enabled better understanding of heterogeneity within in vitro and in vivo tissues. Finally, we provide a few work-arounds for adapting current on-chip culture methods to better mimic physiological cell heterogeneity including accounting for crucial rare cell types and events.