Capturing nascent extracellular vesicles by metabolic glycan labeling-assisted microfluidics.

Extracellular vesicle (EV) secretion is a dynamic process crucial to cellular communication. Temporally sorting EVs, i.e., separating the newly-produced ones from the pre-existing, can allow not only deep understanding of EV dynamics, but also the discovery of potential EV biomarkers that are related to disease progression or responsible to drug intervention. However, the high similarity between the nascent and pre-existing EVs makes temporal separation extremely challenging. Here, by co-translational introduction of azido groups to act as a timestamp for click chemistry labelling, we develop a microfluidic-based strategy to enable selective isolation of nascent EVs stimulated by an external cue. In two mouse models of anti-PD-L1 immunotherapy, we demonstrate the strategy's feasibility and reveal the high positive correlation of nascent PD-L1+ EV level to tumor volume, suggesting an important role of nascent EVs in response to immunotherapy in cancer treatment.

Reliable Detection of Extracellular PD-L1 by DNA Computation-Mediated Microfluidics.

Extracellular vesicle PD-L1 (programmed death-1 ligand 1) is of greater value in tumor diagnosis, prognosis, and efficacy monitoring of anti-PD-1/PD-L1 immunotherapy. However, soluble PD-L1 interferes with the accurate detection of extracellular vesicle (EV) PD-L1. Here, we developed a microfluidic differentiation method for the detection of extracellular PD-L1, without the interference of soluble, by DNA computation with lipid probes and PD-L1 aptamer as inputs (DECLA). For the developed DECLA method, a cholesterol-DNA probe was designed that efficiently embeds into the EV membrane, and an aptamer-based PD-L1 probe was used for PD-L1 recognition. Due to the stable secondary structure of the designed connector, only cobinding of cholesterol-DNA and PD-L1 affinity probe induced biotin-labeled connector activation, while soluble PD-L1 cannot hybridize. As a result, PD-L1 EVs can be efficiently captured by streptavidin-functioned herringbone chip and quantified by anti-CD63-induced fluorescence signal. The high specificity of dual-input DNA computation allied to the high sensitivity of microfluidic-based detection was suitable for distinguishing lung cancer patients from healthy donors, highlighting its potential translation to clinical diagnosis and therapy monitoring.

Isolation of PD-L1 Extracellular Vesicle Subpopulations Using DNA Computation Mediated Microfluidic Tandem Separation.

Accurate isolation of targeted extracellular vesicle (EV) is challenging due to the antigenic heterogeneity of EV subpopulations which are from different cell origins. Most EV subpopulations lack a single marker whose expression cleanly distinguishes them from mixed populations of closely related EVs. Here, a modular platform capable of taking multiple binding events as input, performing logic computations, and producing two independent outputs for tandem microchips for EV subpopulation isolation, is developed. Taking advantages of the excellent selectivity of dual-aptamer recognition and the sensitivity of tandem microchips, this method achieves, for the first time, sequential isolation of tumor PD-L1 EVs and non-tumor PD-L1 EVs. As a result, the developed platform can not only effectively distinguish cancer patients from healthy donors but also provides new clues for assessing immune heterogeneity. Moreover, the captured EVs can be released through a DNA hydrolysis reaction with high efficiency, which is compatible with downstream mass spectrometry for EV proteome profiling. Overall, this strategy is expected to isolate different EV subpopulations, translate EVs into reliable clinical biomarkers, and accurately investigate the biological functions of different EV subsets.

Aptamer-LYTACs for Targeted Degradation of Extracellular and Membrane Proteins.

Recently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N-acetylgalactosamine (tri-GalNAc) is a complex and time-consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer-based LYTAC (Apt-LYTAC) to realize liver-cell-specific degradation of extracellular and membrane proteins by conjugating aptamers to tri-GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt-LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt-LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

Magnetofluid-Integrated Multicolor Immunochip for Visual Analysis of Neutralizing Antibodies to SARS-CoV-2 Variants.

The global spread of SARS-CoV-2 virus has severely affected human health, life, and work. Vaccine immunization is considered to be an effective means to protect the body from infection. Therefore, timely analysis of the antibody level is helpful to identify people with low immune response or attenuated antibodies so as to carry out targeted and precise vaccine booster immunization. Herein, we develop a magnetofluid-integrated multicolor immunochip, as a sample-to-answer system in a fully enclosed space, for visual analysis of neutralizing antibodies of SARS-CoV-2. Generally, this chip adopts an innovative three-dimensional two-phase system that utilizes mineral oil to block the connection between reagent wells in the vertical direction and provides a wide interface for rapid and nondestructive shuttle of magnetic beads during the immunoassay. In order to obtain visualized signal output, gold nanorods with a size-dependent color effect are used as the colorful chromogenic substrates for evaluation of the antibody level. Using this chip, the neutralizing antibodies were successfully detected in vaccine-immunized volunteers with 83.3% sensitivity and 100% specificity. Furthermore, changes in antibody levels of the same individual over time were also reflected by the multicolor assay. Overall, benefiting from simple operation, airtight safety, and nonrequirement of external equipment, this platform can provide a new point-of-care testing strategy for alleviating the shortage of medical resources and promoting epidemic control in underdeveloped areas.

Tracing Tumor-Derived Exosomal PD-L1 by Dual-Aptamer Activated Proximity-Induced Droplet Digital PCR.

Tumor-derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell-derived exosomes. Herein, we developed a dual-target-specific aptamer recognition activated in situ connection system on exosome membrane combined with droplet digital PCR (ddPCR) (TRACER) for quantitation of tumor-derived exosomal PD-L1 (Exo-PD-L1 ). Leveraging the high binding affinity of aptamers, excellent selectivity of dual-aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor-derived Exo-PD-L1 in a wash-free manner. Due to the excellent sensitivity, the level of tumor-derived Exo-PD-L1 detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo-PD-L1 . The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.

Preparation and characterization of citric acid crosslinked konjac glucomannan/surface deacetylated chitin nanofibers bionanocomposite film.

Novel bionanocomposite films were prepared by combining konjac glucomannan/surface deacetylated chitin nanofibers (KGM/S-ChNFs) with different concentrations of citric acid (CA) (10%-25%) via a solution casting method. The effect of CA-induced crosslinking on the rheological behavior of film-forming solutions (FFS) as well as the structural and physicochemical properties of the resulting bionanocomposite films were evaluated. The results revealed that the increased CA loadings increased the shear viscosity of FFS. Fourier transform infrared spectra and scanning electron microscopy results confirmed the successful crosslinking between CA and S-ChNFs. The addition of 20 wt% CA was defined as the optimal condition, resulting in minimum water sensitivity and permeability, while maintaining a good combination of tensile strength and antimicrobial properties. This work supported the conclusion that CA crosslinking was an effective pathway for the preparation of polysaccharide-based bionanocomposite films with improved properties, which may be a promising material for active food packaging applications.

Transparent bionanocomposite films based on konjac glucomannan, chitosan, and TEMPO-oxidized chitin nanocrystals with enhanced mechanical and barrier properties.

The development of biopolymer-based films for food packaging is increasing owing to their environmental appeal, renewability, and biodegradability. In this study, transparent and biodegradable konjac glucomannan (KGM)/chitosan (CS)/TEMPO-oxidized chitin nanocrystal (TEMPO-ChNCs) bionanocomposite films were prepared. The TEMPO-ChNCs were prepared from chitin using the 2,2,6,6-tetramethylpiperidine-1-oxylradical (TEMPO) oxidation method and were used as a reinforcement nanofiller for the bionanocomposite films. The effect of TEMPO-ChNCs content on both rheological properties of film-forming solutions (FFS) and structural and physical properties of the resultant films was investigated. The rheological results of the FFS revealed that the TEMPO-ChNCs interacted with KGM and CS through electrostatic interaction and the hydrogen bonds in the bionanocomposite matrix, which was in agreement with the Fourier transform infrared spectroscopy and X-ray diffraction results. The microstructure of the films showed that 3% (w/w) TEMPO-ChNCs were homogeneously dispersed within the KGM/CS matrix, reducing the free volume of the biocomposite matrix and improving the final film mechanical and barrier properties (P < 0.05). Furthermore, these bionanocomposite films exhibited good thermal stability. The incorporation of TEMPO-ChNCs in the KGM/CS matrix produced flexible and transparent bionanocomposite films. Thus, this bionanocomposite films has potential use in food packaging applications.

In situ self-assembly chitosan/ε-polylysine bionanocomposite film with enhanced antimicrobial properties for food packaging.

A set of chitosan/ε-polylysine (ε-PL) bionanocomposite films were prepared by a simple in situ self-assembly technique using sodium tripolyphosphate (TPP) as cross-linking agent. The physical, mechanical, structural, and antimicrobial properties of these films were investigated. Fourier infrared spectroscopy and X-ray diffraction showed that the introduction of TPP promoted the formation of hydrogen bonds and electrostatic interactions among functional groups of chitosan or ε-PL, which improved the tensile strength and decreased the water solubility, water vapor permeability and surface wettability of films. On the other hand, the incorporation of ε-PL weakened the bionanocomposite film' structure and integrity, resulting in a decrease trend of films' mechanical and barrier properties. More importantly, the bionanocomposite films exhibited excellent antimicrobial efficacy against E. coli and S. aureus by the increasing ratio of ε-PL. And ε-PL presented a sustained release from the films, which was closely related to TPP concentration. Results of this study suggested that chitosan/ε-PL films could be used as antimicrobial bio-material and have great potential in food industry.

[Effects of nitroglycerin on plasma calcitonin gene-related peptide and endothelin in congestive heart failure].

OBJECTIVE: To investigate the plasma calcitonin gene-related peptide (CGRP) and endothelin (ET) levels and the effects of nitroglycerin on CGRP and ET in congestive heart failure (CHF), and to make clear the mechanism of nitroglycerin in vasodilation and cardiac protection. METHODS: The fast blood samples were taken from the peripheral vein of 40 volunteers and 40 CHF patients to estimate the basal level of CGRP and ET. Infusion of nitroglycerin of 25 to 50 micrograms/min started in the forearm vein of CHF patients. Blood samples were taken at the end of the 1 hour infusion and (12 +/- 2) days of nitroglycerin treatment. Plasma concentrations of CGRP and ET were measured by radioimmunoassay. RESULTS: In patients with CHF before the treatment, the plasma concentration of CGRP was significantly lower than that in the controls (P < 0.001), but the concentration of ET significantly higher than that in the controls (P < 0.001). During the nitroglycerin treatment, the plasma concentration of CGRP significantly increased (P < 0.001), whereas the concentration of ET significantly decreased (P < 0.01). The mean arterial pressure significantly decreased (P < 0.001). CONCLUSION: Nitroglycerin can increase the peripheral plasma CGRP level and decrease the concentration of ET when treating CHF. Intravenous nitroglycerin may be beneficial to the improvement of abnormal haemodynamics and neurohormonal derangement.