Protein nanopore reveals the renin-angiotensin system crosstalk with single-amino-acid resolution.

The discovery of crosstalk effects on the renin-angiotensin system (RAS) is limited by the lack of approaches to quantitatively monitor, in real time, multiple components with subtle differences and short half-lives. Here we report a nanopore framework to quantitatively determine the effect of the hidden crosstalk between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) on RAS. By developing an engineered aerolysin nanopore capable of single-amino-acid resolution, we show that the ACE can be selectively inhibited by ACE2 to prevent cleavage of angiotensin I, even when the concentration of ACE is more than 30-fold higher than that of ACE2. We also show that the activity of ACE2 for cleaving angiotensin peptides is clearly suppressed by the spike protein of SARS-CoV-2. This leads to the relaxation of ACE and the increased probability of accumulation of the principal effector angiotensin II. The spike protein of the SARS-CoV-2 Delta variant is demonstrated to have a much greater impact on the crosstalk than the wild type.

Silver-amplified fluorescence immunoassay via aggregation-induced emission for detection of disease biomarker.

Development of simple, robust, and reliable detection strategy of disease biomarkers holds tremendous promise for early clinical diagnosis and prognosis of diseases. In this work, through combining a silver nanoparticle (AgNP) linked immunoassay and aggregation induced emission (AIE)-based fluorogenic Ag+ probe, we developed a silver-amplified fluorescence immunoassay for the detection of disease biomarkers. This method overcame the intrinsic limitations of enzymes as the dissolution of AgNPs generated numerous Ag+, which could switch on the fluorogenic Ag+ probe driven by tetrazolate-Ag+ complexation. As a proof of concept, our method could be used for determining α-fetoprotein (AFP) with a linear relationship in concentrations ranging from 0.1 ng mL-1 to 5 µg mL-1 and a low limit of detection of 42 pg mL-1. Our method was successfully confirmed for the detection of AFP in real serum samples from hepatocellular carcinoma (HCC) patients, demonstrating the great potential for clinical diagnosis.

Plasmon-Induced Photoreduction System Allows Ultrasensitive Detection of Disease Biomarkers by Silver-Mediated Immunoassay.

Current strategies for the detection of disease biomarkers often require enzymatic assays that may have limited sensitivity due to inferior stability and vulnerable catalytic activity of the enzyme. A new enzyme-free amplification method for identifying suitable biomarkers is necessary to lower the limit of detection and improve many critical diagnosis applications. Here, we presented an enzyme-free amplified plasmonic immunoassay that enhanced the detection sensitivity of disease biomarkers by combining a novel plasmon-induced silver photoreduction system with a silver nanoparticle (AgNP)-linked immunoassay. The key step to achieving ultrasensitivity was to use Ag+ from dissolved AgNPs that control the growth rate of the silver coating on plasmonic nanosensors under visible light illumination. We demonstrated the outstanding sensitivity and robustness of this assay by detecting the disease biomarker alpha-fetoprotein (AFP) at a low concentration of 3.3 fg mL-1. The detection of AFP was further confirmed in the sera of hepatocellular carcinoma patients.

Enzyme-free amplified SERS immunoassay for the ultrasensitive detection of disease biomarkers.

We developed a novel enzyme-free amplified SERS immunoassay by combining silver nanoparticle (AgNP)-linked immunoreaction and SERS transduction for the detection of disease biomarkers. As a proof of concept, our method was successfully illustrated with the disease biomarker α-fetoprotein with the detection limit of 3.3 × 10-13 g mL-1 and a double-blind experiment consisting of tens of serum samples was performed to confirm its reliability.

Sensitive detection of protein biomarkers using silver nanoparticles enhanced immunofluorescence assay.

Detection of biomarkers is extremely important for the early diagnosis of diseases. Here, we developed an easy and highly sensitive fluorescence detection system for the determination of biomarkers by combining the rapid separation of magnetic beads and silver nanoparticles labeled antibodies. An ultrasensitive silver ions fluorescence probe 3', 6'-bis (diethylamino)-2-(2-iodoethyl) spiro[isoindoline-1, 9'-xanthen]-3-one (Ag+-FP) was applied to immunoassay. A significant signal amplification was achieved as the AgNPs can be dissolved by H2O2 and generate numerous Ag+, which would turn "on" the fluorescence of Ag+-FP. Using α-fetoprotein (AFP) and C-reactive protein (CRP) as target analytes, good linear responses were obtained from 0.1 to 10 ng mL-1 and the limits of detection (LOD) were as low as 70 pg·mL-1 and 30 pg·mL-1, respectively. In addition, the developed system was further evaluated for the detection of real samples including 30 positive serum specimens obtained from hepatocarcinoma patients and 20 negative serum samples, and performs as well as the commercial electrochemiluminescence immunoassay (ECLI) method with less cost and more convenience. Thus, the designed detection system can be used as a promising platform for the detection of a variety of biomarkers and served as a powerful tool in clinical diagnosis.

Metal-linked Immunosorbent Assay (MeLISA): the Enzyme-Free Alternative to ELISA for Biomarker Detection in Serum.

Determination of disease biomarkers in clinical samples is of crucial significance for disease monitoring and public health. The dominating format is enzyme-linked immunosorbent assay (ELISA), which subtly exploits both the antigen-antibody reaction and biocatalytic property of enzymes. Although enzymes play an important role in this platform, they generally suffer from inferior stability and less tolerant of temperature, pH condition compared with general chemical product. Here, we demonstrate a metal-linked immunosorbent assay (MeLISA) based on a robust signal amplification mechanism that faithfully replaces the essential element of the enzyme. As an enzyme-free alternative to ELISA, this methodology works by the detection of α-fetoprotein (AFP), prostatic specific antigen (PSA) and C-reactive protein (CRP) at concentrations of 0.1 ng mL(-1), 0.1 ng mL(-1) and 1 ng mL(-1) respectively. It exhibits approximately two magnitudes higher sensitivity and is 4 times faster for chromogenic reaction than ELISA. The detection of AFP and PSA was further confirmed by over a hundred serum samples from hepatocellular carcinoma (HCC) and prostate cancer patients respectively.

[Effects of Poly I:C in inducing growth inhibition and apoptosis of human hepatocellular carcinoma cells].

OBJECTIVE: To explore the effect and mechanism of Poly I:C in inducing growth inhibition and apoptosis of human hepatocellular carcinoma SMMC-7721 cells. METHODS: SMMC-7721 cells were treated with different doses of Poly I:C for 24, 48, and 72 h, and the cell growth inhibition rate was analyzed with CCK-8 assay. The cell cycle and the apoptosis were analyzed using flow cytometry with Annexin-V and PI staining, and quantitative RT-PCR analysis were used to detect the expression of TLR3, TRIF, and IFN-beta mRNA in cells. RESULTS: In the cells exposed to Poly I:C at low, moderate, and high doses, the inhibitory rates was the highest in high-dose Poly I:C group, and at a given Poly I:C dose, prolonged exposure resulted in significantly increased cell growth inhibition rate (P<0.05). Flow cytometry showed that Poly I:C induced cell apoptosis in a time- and dose-dependent manner and significantly increased the percentage of G1-phase cells as compared with that in the control group. The mRNA level of TLR3, TRIF, and IFN-beta were also increased following Poly I:C treatment in comparison with the control group. CONCLUSION: Poly I:C can induce significant growth inhibition and apoptosis of SMMC-7721 cells in a dose- and time-dependent manner possibly by causing cell cycle arrest and TLR3 signaling pathway activation that leads to IFN-beta production and cell apoptosis.

Expression of ubiquitin-conjugating enzyme E2C/UbcH10 in astrocytic tumors.

UbcH10 is one of the key regulators of cell cycle progression through the mitotic spindle assembly checkpoint pathway. Recently, aberrantly high UbcH10 expression has been demonstrated in a variety of malignancies. However, its role in astrocytic carcinogenesis is not well defined. This study investigated the splice pattern of the UbcH10 gene and its expression status in astrocytomas of different grades. Consequently, UbcH10 splice variant 1 (GenBank accession nos. NM_007019) was detected in astrocytomas and normal brain tissues by RT-PCR and sequence analysis. Expression levels of UbcH10 mRNA were elevated in high- versus low-grade astrocytomas (64.33+/-60.98 vs 8.36+/-8.15, respectively; p=0.000) or normal controls (64.33+/-60.98 vs 1.00+/-1.57, respectively; p=0.000), as determined by quantitative real time PCR analysis. Similarly, immunohistochemistry study showed increased UbcH10 labelling index in high-grade astrocytomas versus low-grade tumors (10.53+/-5.79% vs 4.23+/-2.85%, respectively; p=0.000) or normal controls (10.53+/-5.79% vs 0.0+/-0.0%, respectively; p=0.000) and, a positive correlation between UbcH10 immunoreactivity and Ki-67 immunostaining was also noted (Spearman r=0.63, p<0.001). These data suggest that overexpression of UbcH10 may serve as one important molecular mechanism that underlies the astrocytic carcinogenesis.

[The preparation of human anti-HBs Fab fragment by bioengineering technique].

AIM: To prepare human anti-HBs Fab by bioengineering technique. METHODS: The specific human anti-HBs Fab gene screened from combinatorial library was cloned into plasmid pBAD/g IIIA, then positive clone was transformed into E.coli Top10. After the fermentation and expression processes, the soluble Fab fragment in the periplasm were purified by Ni-NTA-agarose affinity chromatography,and the inclusion bodies were in turn denatured, solubilized, purified and renatured. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. RESULTS: The quantity of soluble Fab protein purified from periplasm with Ni-NTA-Agarose which possessed good specificity as well as excellent binding activity to antigens was up to 80 mg per liter, but the biologically active protein acquired after renaturation of the inclusion bodies was quite small. CONCLUSION: Using pBAD/g IIIA-Top10 expression system, the soluble Fab protein with biological activity could be produced from periplasm of the E.coli Top10, and the strategy is available to prepare human anti-HBsAg Fab fragments in large quantity by gene engineering technique.

[Fed-batch fermentation of Escherichia coli that express fab fragment of anti-HBsAg].

To develop a fed-batch fementation process of E. coli TOP10 containing a recombinant plasmid pBAD/HBs Fab. Cells were grown in semi-defined medium at 37 degrees C, and the feed operation using glycerol as carbon source was performed when dissolved oxygen increased. When the target cell concentration reached to 64g/L, arabinose was added to a final concentration of 0.02%. Cells were grown for another 5h with the culture temperature decreased from 37 degrees C to 30 degrees C. In the whole process, cell growth was monitored by measuring OD600 of samples taken at 1/2h intervals and the dissolved oxygen was kept above 30%. After the fementation, E. coli pellets were collected for purification of Fab protein. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. Cell concentration we got is 96g wet bacteria per liter, the Fab protein is about 6% of total protein of the host, that is 80mg per liter. Stable fermentation parameters were obtained for fermentation to improve productivity of the Fab protein. The Fab protein was produced in the form of soluble biologically active protein, it's better than inclusion bodies from which biologically active protein can only be recovered by complicated and costly denaturation and refolding process.