Building Dynamic Cellular Machineries in Droplet-Based Artificial Cells with Single-Droplet Tracking and Analysis.

Although the application of droplet microfluidics has grown exponentially in chemistry and biology over the past decades, robust universal platforms for the routine generation and comprehensive analysis of droplet-based artificial cells are still rare. Here we report using microfluidic droplets to reproduce a variety of types of cellular machinery in in vitro artificial cells. In combination with a unique image-based analysis method, the system enables full automation in tracking single droplets with high accuracy, high throughput, and high sensitivity. These powerful performances allow broad applicability evident in three representative droplet-based analytical prototypes that we develop for (i) droplet digital detection, (ii) in vitro transcription and translation reactions, and (iii) spatiotemporal dynamics of cell-cycle oscillations. The capacities of this platform to generate, incubate, track, and analyze individual microdroplets via real-time, long-term imaging unleash its great potential in accelerating cell-free synthetic biology. Moreover, the wide scope covering from digital to analog to morphological detections makes this droplet analysis technique adaptable for many other divergent types of droplet-based chemical and biological assays.

Peptide-Lipid Interaction Sites Affect Vesicles' Responses to Antimicrobial Peptides.

This article presents coarse-grained molecular dynamics simulations of pore-forming antimicrobial peptide melittin and its interactions with vesicles composed of a mixture of zwitterionic and anionic phospholipids. Besides creating holes in the membrane, the adsorption of melittin also induces vesicle budding, which can develop into vesiculation at high peptide concentrations, as well as vesicle invagination, which can eventually result in a corrugated membrane surface. These rich morphology changes are mediated by the curvature of the vesicles and the peptide concentration. Highly curved vesicles favor the recruitment of melittins with a higher density of binding sites. The peptides mainly penetrate into the membrane surface in monomers via hydrophobic interaction. Lowly curved vesicles recruit melittins with a low density of binding sites. Surplus peptides are prone to form oligomers and shallowly adsorb on the surface of membrane via electrostatic interaction. The penetration of monomers induces membrane pore formation and positive membrane curvature, which promote vesicle budding. The adsorption of oligomers induces negative membrane curvature, which promotes vesicle invagination. This work demonstrates that antimicrobial peptides adopt multiple actions to destroy bacterial membranes.

2-Year Efficacy, Immunogenicity, and Safety of Vigoo Enterovirus 71 Vaccine in Healthy Chinese Children: A Randomized Open-Label Study.

BACKGROUND: This study evaluated the 2-year efficacy, immunogenicity, and safety of the Vigoo enterovirus 71 (EV71) vaccine. METHOD: In an initial phase 3 study, we randomly assigned healthy infants and children aged 6-35 months (ratio, 1:1) to receive 2 doses of either EV71 vaccine (5120 participants) or placebo (5125 participants) at days 0 and 28, and followed them for 12 months after vaccination. In this extended follow-up study, we continued to evaluate the efficacy, immunogenicity, and safety of the EV71 vaccine for up to 2 years. RESULTS: Overall efficacy was 94.84% (95% confidence interval [CI], 83.53%-98.38%) during the 2-year follow-up period (P < .0001), and the vaccine efficacy during the second year was 100.00% (95% CI, 84.15%-100.00%) against EV71-associated hand-foot-and-mouth disease (HFMD; P < .0001). Geometric mean titers of neutralizing antibody in participants remained high during the 2-year follow-up period, and no vaccine-related serious adverse events were recorded. CONCLUSIONS: Two doses of Vigoo EV71 vaccine could provide sustained protection against EV71-associated HFMD in healthy Chinese children. CLINICAL TRIALS REGISTRATION: NCT01508247.

Microplastics induce neurotoxicity in aquatic animals at environmentally realistic concentrations: A meta-analysis.

Microplastics (MPs) draw international attention owing to their widespread distribution in water ecosystems, but whether MPs cause neurotoxic effects in aquatic animals at environmentally realistic concentrations is still controversial. This meta-analysis recompiled 35 studies to determine whether MPs could change the levels of brain (in vivo) neurotransmitters in aquatic animals at environmentally realistic concentrations (≤1 mg/L, median = 0.100 mg/L). Then, a group comparison was conducted to compare the effects of different factors on the effect size and to explore the significant factors affecting the neurotoxicity of MPs. The results demonstrated that MP exposure could considerably decrease the levels of acetylcholinesterase (AchE) in the brain of aquatic animals by 16.2%. However, the effects of MPs on cholinesterase (CHE), acetylcholine (ACh), dopamine (DA) and γ-aminobutyric acid (GABA) were not statistically significant due to the small number of studies and samples. The neurotoxicity of MPs was closely linked with particle size and exposure time but independent of animal species, MP compositions, MP morphology and MP concentrations. Further literatures review indicated that MP-induced neurotoxicity and behavioral changes are related with multiple biological processes, including nerve damage, oxidative stress, intestinal flora disturbance and metabolic disorder. Furthermore, some factors influencing MP neurotoxicity in the real environment (e.g. the aging of MPs, the release of MP additives, and the co-exposure of MPs and pollutants) were discussed. Overall, this study preliminarily explored whether MPs induced changes in neurotoxicity-related indicators in aquatic animals through meta-analysis and provided scientific evidence for evaluating the health risks and neurotoxicity of MPs at the environmental level.

A cyanoacrylate/triethyl citrate/nanosilica-based closure glue with wet-adhesion capability for treatment of superficial varicose veins.

Varicose veins in legs are common in clinics. Currently, medical adhesive-based, minimally invasive endovenous occlusion is used to treat them. However, the most common cyanoacrylate medical adhesives do not perform well when used under blood/wet conditions. In particular, poor adhesion, short curing time, and high heat release greatly limit their clinical use. In this paper, we demonstrate the use of a composite system composed of butyl-cyanoacrylate, triethyl citrate, and nanosilica that exhibits a blood/wet-adhesion capability to serve as a new sealing glue. Hydrophobic triethyl citrate groups displace boundary waters while also protecting cyanoacrylate monomers from undergoing rapid polymerization. Nanosilica increases viscosity, which contributes to in situ extrusion molding and retention. An optimal formulation, FAL-006, exhibited good physical and chemical properties in vitro. The performed additional safety assays indicated that FAL-006 has good biocompatibility. The closure efficiency of FAL-006 in vivo was evaluated in both a rat abdominal aortic closure model and in a sheep lower limb venous closure model. Taken together, these results indicate that FAL-006 exhibits promising potential for use in clinical applications. Furthermore, this study provides a new strategy for designing underwater adhesive agents for additional clinical applications, and a strategy for constructing other biomaterials needed for use under wet conditions.

A contact-polymerizable hemostatic powder for rapid hemostasis.

The immediate control of a hemorrhage is crucial for reducing fatalities in critical situations such as battlefields, traffic accidents, natural disasters, etc. Most existing commercial hemostatic powders have weak adhesion capability and poor biodegradability, restricting their clinical use. In this paper, a new poly(ethylene glycol)-di(cyanoacrylate) (CA-PEG-CA)-based hemostatic powder with tissue-contact-triggered strong adhesion and controlled fast degradation is proposed. The monomers quickly underwent crosslinking polymerization while in contact with tissue or blood, forming an in situ gel on the wound. The hemostatic mechanism was demonstrated to depend on both adhesive-based sealing and the aggregation of platelets and erythrocytes. The powder showed excellent hemostatic effects both in vitro and in vivo, even in a rat model with a weakened native hemostatic capacity. In addition, the poly-CA-PEG-CA gel could be rapidly biodegraded by ester bond hydrolysis. Notably, a cysteamine (CS)-containing solution could accelerate the degradation rate, endowing the gel with an on-demand removal property. This hemostatic powder not only can be used to efficiently control bleeding in emergency scenarios, but it can also allow nontraumatic re-exposure of wounds during subsequent surgical care. These properties make the CA-PEG-CA powder a promising candidate to act as a multifunctional wound care agent for first aid.

A robust and tunable mitotic oscillator in artificial cells.

Single-cell analysis is pivotal to deciphering complex phenomena like heterogeneity, bistability, and asynchronous oscillations, where a population ensemble cannot represent individual behaviors. Bulk cell-free systems, despite having unique advantages of manipulation and characterization of biochemical networks, lack the essential single-cell information to understand a class of out-of-steady-state dynamics including cell cycles. Here, by encapsulating Xenopus egg extracts in water-in-oil microemulsions, we developed artificial cells that are adjustable in sizes and periods, sustain mitotic oscillations for over 30 cycles, and function in forms from the simplest cytoplasmic-only to the more complicated ones involving nuclear dynamics, mimicking real cells. Such innate flexibility and robustness make it key to studying clock properties like tunability and stochasticity. Our results also highlight energy as an important regulator of cell cycles. We demonstrate a simple, powerful, and likely generalizable strategy of integrating strengths of single-cell approaches into conventional in vitro systems to study complex clock functions.

Short-term intratracheal use of PEG-modified IL-2 and glucocorticoid persistently alleviates asthma in a mouse model.

Regulatory T (Treg) cells play an important role in allergic airway diseases, and upregulation of Treg cells is a potential therapeutic strategy for asthma. In this study, we show that short-term intratracheal use of IL-2 combined with glucocorticoid alleviates antigen-induced airway inflammation and reduces airway hyperresponsiveness by expanding antigen-nonspecific Treg cells, with a decrease in T helper 2 (Th2) cells and Th2-associated cytokines. We also designed a long-acting polyethylene glycol (PEG)-modified IL-2 and demonstrated that the optimal dosage form is IL-2(PEG) plus budesonide, which can upregulate Treg cells and ameliorate asthma at a lower dose. The therapeutic effect was faster than treatment with dexamethasone and was effective at a low dose suitable for humans that could last for at least 6 weeks. This study unveils a new therapeutic regimen and suggests that such endogenous Treg therapy could be a useful tool to persistently alleviate asthma.