Integrated microfluidic system for isolating exosome and analyzing protein marker PD-L1.

Exosomes are lipid-bilayered nanovesicles secreted by cells to mediate intercellular communication. Various kinds of biomolecules involved in exosomes offer non-invasive approaches for detecting or monitoring disease and developing targeted therapeutics. Here, we present an integrated microfluidic exosome isolation and detection system (EXID system) to analyze the abundance of the exosomal PD-L1 protein marker, which is a transmembrane protein expressed by tumors to suppress immune activation of T cells. By incorporating exosome isolation and biomarker labelling and quantification within a single microfluidic chip, our system reduced the total analysis time below 2 h. Using the EXID system, 7 categories of cell lines including cancer cell lines and control samples were profiled, where significant differences in the fluorescence intensity were observed with the limit of detection (LOD) down to 10.76 per microliter. Such noticeable variations in PD-L1 abundance among cancer cell lines highlighted the need of personalized treatments. Furthermore, 16 clinical samples from 7 post-treated cancer patients, 3 prior-treatment patients and 6 healthy controls, are tested, among which differences in sensitivity toward immune response were subsistent. Because the abundance of PD-L1 reflects the sensibility for immune response, our results provide useful guides to design immunotherapy strategies for different types of tumors.

Ultrafast multiplexed detection of SARS-CoV-2 RNA using a rapid droplet digital PCR system.

We report the first combination of droplet digital and rapid PCR techniques for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. The presented rapid digital PCR system simultaneously detects two specific targets (ORF1ab and N genes) and one reference gene (RNase P) with a single PCR thermal cycling period around 7 s and the total running time less than 5 min. A clear positive signal could be identified within 115 s via the rapid digital RT-PCR, suggesting its efficiency for the end-point detection. In addition, benchmark tests with serial diluted reference samples of SARS-CoV-2 RNA reveal the excellent accuracy of our system (R2>0.99). More importantly, the rapid digital PCR system gives consistent and accurate detection of low-concentration reference samples, whereas qPCR yields Ct values with significant variations that could lead to false-negative results. Finally, we apply the rapid digital PCR system to analyze clinical samples with both positive and control cases, where results are consistent with qPCR test outcomes. By providing similar accuracy with qPCR while minimizing the detection time-consuming and the false-negative tendency, the presented rapid digital PCR system represents a promising improvement on the rapid diagnosis of COVID-19.

Characterization and bioactivity of grass carp (Ctenopharyngodon idella) interleukin-21: Inducible production and involvement in inflammatory regulation.

In mammals, interleukin 21 (IL-21) is a broad pleiotropic cytokine that plays critical roles in the development of several inflammatory and autoimmune diseases. In fish, functional information of Il-21 is limited, and its role in immune response is largely unknown. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) il21 gene (gcil21). To characterize the release patterns and biological activity of gcIl-21, we prepared recombinant gcIl-21 (rgcIl-21) and obtained the polyclonal antibody with gcIl-21 specificity. Western blotting analysis showed that in grass carp head kidney leukocytes (HKLs), gcIl-21 was undetected in culture supernatant of untreated cells but drastically induced by heat-killed Aeromonas hydrophila (A. hydrophila), uncovering the release features of gcIl-21 and its possible involvement in immune response. Subsequent functional experiments revealed that rgcIl-21 did not affect the mRNA expression of grass carp il1b and tgfb, but induced a strong expression of grass carp il10, and to a lesser extent of grass carp tnfa in HKLs, suggesting a dominant effect of gcIl-21 in modulating Il-10 signaling as seen in rainbow trout and mammals. Furthermore, in vivo studies showed that intraperitoneal injection of rgcIl-21 was able to increase the survival rate of grass carp infected with live A. hydrophila, and reduce the pathological responses caused by the same pathogenic bacteria in head kidney and intestine. Taken together, these results for the first time revealed the close relationship of fish Il-21 production and function with inflammatory responses, and highlighted its anti-bacterial and anti-inflammatory ability, thereby providing a new insight into host defense mechanisms in fish.

In vitro characterization of grass carp (Ctenopharyngodon idella) IL-26 in regulating inflammatory factors.

Interleukin 26 (IL-26) gene has been identified in human, amphibian and teleost but not in rodents. It is well accepted that IL-26 was a crucial member of IL-10 family which acts as a pro-inflammatory cytokine in human. However, the role of IL-26 in regulating inflammation in lower vertebrates including teleost has not been defined yet. In the present study, grass carp IL-26 (gcIL-26) coding sequence was isolated and identified. Its chromosomal synteny was also analyzed, showing that gcIL-26 gene is flanked by IL-22 and IFN-γ genes with the same transcriptional orientation as seen in human, amphibian and zebrafish. Given that zebrafish and grass carp IL-26 shared relatively low amino acid identities with human IL-26, the functional roles of fish IL-26 are indispensable to be elucidated. Accordingly, recombinant gcIL-26 (rgcIL-26) was prepared by using Pichia pastoris expression system, and it was found to be partially glycosylated. Using grass carp head kidney leucocytes as cell model, rgcIL-26 displayed the bioactivity to stimulate the mRNA expression of some pro-inflammatory cytokines including IL-8, IL-1ß and IL-6, while inhibit mRNA expression of an anti-inflammatory cytokine, IL-10. Moreover, rgcIL-26 also up-regulated inos expression and NO production in grass carp monocytes/macrophages, strengthening its pro-inflammatory properties in fish. Those results collectively demonstrated the functional role of IL-26 in regulating inflammatory response in fish.