We still know very little about how the human immune system responds to SARS-CoV-2. Here we construct a SARS-CoV-2 proteome microarray containing 18 out of the 28 predicted proteins and apply it to ...the characterization of the IgG and IgM antibodies responses in the sera from 29 convalescent patients. We find that all these patients had IgG and IgM antibodies that specifically bind SARS-CoV-2 proteins, particularly the N protein and S1 protein. Besides these proteins, significant antibody responses to ORF9b and NSP5 are also identified. We show that the S1 specific IgG signal positively correlates with age and the level of lactate dehydrogenase (LDH) and negatively correlates with lymphocyte percentage. Overall, this study presents a systemic view of the SARS-CoV-2 specific IgG and IgM responses and provides insights to aid the development of effective diagnostic, therapeutic and vaccination strategies.
The ongoing global novel coronavirus pneumonia COVID‐19 outbreak has engendered numerous cases of infection and death. COVID‐19 diagnosis relies upon nucleic acid detection; however, currently ...recommended methods exhibit high false‐negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long‐read, real‐time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS‐CoV‐2 and other respiratory viruses simultaneously within 6–10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS‐CoV‐2 reaches 100%. Parallel testing with approved real‐time reverse transcription‐polymerase chain reaction kits for SARS‐CoV‐2 and NTS using 61 nucleic acid samples from suspected COVID‐19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS‐CoV‐2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID‐19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.
A detection technology, nanopore targeted sequencing (NTS), for the accurate and comprehensive detection of SARS‐CoV‐2 and other respiratory viruses within 6–10 h is developed, which is suitable for the identification of suspected cases and used as a supplementary technique for the SARS‐CoV‐2 test. NTS can also monitor mutations in the virus and the type of virus.
Correction for 'Promoter-regulated
asymmetric self-assembly strategy to synthesize heterogeneous nanoparticles for signal amplification' by Chen Chen
,
, 2022,
, 16180-16184, ...https://doi.org/10.1039/D2NR04661J.
Intracellular protein delivery is highly desirable for protein drug‐based cell therapy. Established technologies suffer from poor cell‐specific cytosolic protein delivery, which hampers the targeting ...therapy of specific cell populations. A fusogenic liposome system enables cytosolic delivery, but its ability of cell‐specific and controllable delivery is quite limited. Inspired by the kinetics of viral fusion, we designed a phosphorothioated DNA coatings‐modified fusogenic liposome to mimic the function of viral hemagglutinin. The macromolecular fusion machine docks cargo‐loaded liposomes at the membrane of target cells, triggers membrane fusion upon pH or UV light stimuli, and facilitates cytosolic protein delivery. Our results showed efficient cell‐targeted delivery of proteins of various sizes and charges, indicating the phosphorothioated DNA plug‐in unit on liposomes could be a general strategy for spatial‐temporally controllable protein delivery both in vitro and in vivo.
Cell‐specific and stimuli‐responsive cytosolic protein delivery was achieved through a macromolecular fusion machine composed of protein‐loading liposomes and fusogenic phosphorothioated DNA coatings, which mimic the recognition and stimuli‐responsive conformational changes of viral hemagglutinin. This fusion machine provides a versatile platform for various cell type‐dependent biomacromolecules delivery both in vitro and in vivo.
Signal amplification is commonly used to enhance the sensitivity of biological analysis. Here, we present a strategy involving
asymmetric self-assembly combined with promoter strength regulation to ...synthesize heterogeneous nanoparticles for signal amplification. Two expression vectors were constructed by genetically inserting, respectively, signal and binding molecules into the hepatitis B core antigen protein (HBcAg) structure. Because of differential expression of the two recombinant proteins in the presence of a strong promoter (T7) and a weak promoter (Tac-1) and spontaneous asymmetric self-assembly
, heterogeneous HBcAg nanoparticles (NPs) with a high ratio of signal-bearing to target-binding molecules were obtained. These nanoparticles contained a large number of green fluorescent proteins as signal molecules and a small number of B1 immunoglobulin-binding domains from protein G for antibody binding, thus enabling sensitive immunoassays. As a proof of concept, improved sensitivity for antibody detection was achieved using the heterogeneous nanoparticle conjugated with a secondary antibody molecule.
Chiral recognition of enantiomers is fundamentally important. In this study, a novel strategy for the chiral discrimination of glucose enantiomers was constructed based on the hydrogen peroxide ...(H2O2)-mediated generation of a carbon dots-gold nanoparticle (C-dots@Au NP) complex independent of surface modification with chiral ligands. H2O2 is essential as a reductant to promote the growth of Au NPs from gold salts. Besides, the modification of C-dots with sulfhydryl groups is necessary for its anchoring on the surface of Au NPs. Therefore, in the presence of H2O2, the C-dots@Au NP complex can be self-generated from a simple mixture containing C-dots and Au salts. It is worth noting that glucose oxidase can selectively catalyze d-glucose but not l-glucose to generate H2O2. In this regard, the chiral recognition process can trigger the formation of the C-dots@Au NP complex. Furthermore, based on the production of reddish Au NPs and the reduction of C-dot fluorescence quenched by Au NPs, the resultant C-dots@Au NP complex enables achieving the chiral discrimination of glucose enantiomers by combining colorimetric and fluorometric assays. Compared with the conventional approaches that use chiral ligands to decorate NPs, the generation of the chiral-responsive C-dots@Au NP complex is much simpler and faster. Upon combination with specific enzymatic reactions that produce a reductive product, the current strategy provides a general approach for the identification of chiral enantiomers.
Since the emergence of SARS-CoV-2, numerous studies have been attempting to determine biomarkers, which could rapidly and efficiently predict COVID-19 severity, however there is lack of consensus on ...a specific one. This retrospective cohort study is a comprehensive analysis of the initial symptoms, comorbidities and laboratory evaluation of patients, diagnosed with COVID-19 in Huoshenshan Hospital, Wuhan, from 4th February to 12th March, 2020. Based on the data collected from 63 severely ill patients from the onset of symptoms till the full recovery or demise, we found not only age (average 70) but also blood indicators as significant risk factors associated with multiple organ failure. The blood indices of all patients showed hepatic, renal, cardiac and hematopoietic dysfunction with imbalanced coagulatory biomarkers. We noticed that the levels of LDH (85%, P < .001), HBDH (76%, P < .001) and CRP (65%, P < .001) were significantly elevated in deceased patients, indicating hepatic impairment. Similarly, increased CK (15%, P = .002), Cre (37%, P = 0.102) and CysC (74%, P = 0.384) indicated renal damage. Cardiac injury was obvious from the significantly elevated level of Myoglobin (52%, P < .01), Troponin-I (65%, P = 0.273) and BNP (50%, P = .787). SARS-CoV-2 disturbs the hemolymphatic system as WBC# (73%, P = .002) and NEUT# (78%, P < .001) were significantly elevated in deceased patients. Likewise, the level of D-dimer (80%, P < .171), PT (87%, P = .031) and TT (57%, P = .053) was elevated, indicating coagulatory imbalances. We identified myoglobin and CRP as specific risk factors related to mortality and highly correlated to organ failure in COVID-19 disease.
This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype ...demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.
The distribution, localization and density of individual molecules (
e.g.
drug-specific receptors) on single cells can offer profound information about cell phenotypes. Profiling this information is ...a new research direction within the field of single cell biology, but it remains technically challenging. Through the combined use of quantum dot labeling, structured illumination microscopy (SIM) and computer-aided local surface reconstruction, we acquired a 3D imaging map of a drug target molecule, integrin αvβ3, on glioblastoma cells at the single cell level. The results revealed that integrin αvβ3 exhibits discrete distribution on the surface of glioblastoma cells, with its density differing significantly among cell lines. The density is illustrated as the approximate number of target molecules per μm
2
on the irregular cell surface, ranging from 0 to 1.6. Functional studies revealed that the sensitivity of glioblastoma cells to inhibitor molecules depends on the density of the target molecules. After inhibitor treatment, the viability and invasion ability of different glioblastoma cells were highly correlated with the density of integrin αvβ3 on their surfaces. This study not only provides a novel protocol for the quantitative analysis of surface proteins from irregular single cells, but also offers a clue for understanding the heterogeneity of tumor cells on the basis of molecular phenotypes. Thus, this work has potential significance in guiding targeted therapies for cancers.
The distribution, localization and density of individual molecules (
e.g.
drug-specific receptors) on single cells can offer profound information about cell phenotypes.
This study presents a microfluidics based cytometry capable of characterizing cell sizes and counting numbers of specific cytosolic proteins where cells were first bound by antibodies labelled with ...fluorescence and then aspirated into a constriction microchannel in which fluorescent levels were measured. These raw fluorescent pulses were further divided into a rising domain, a stable domain and a declining domain. In addition, antibody solutions with labelled fluorescence were aspirated through the constriction microchannel, yielding curves to translate raw fluorescent levels to protein concentrations. By using key parameters of three domains as well as the calibration curves, cell diameters and the absolute number of β-actins at the single-cell level were quantified as 14.2 ± 1.7 μm and 9.62 ± 4.29 × 10
(A549, n
= 14 242), 13.0 ± 2.0 μm and 6.46 ± 3.34 × 10
(Hep G2, n
= 35 932), 13.8 ± 1.9 μm and 1.58 ± 0.90 × 10
(MCF 10 A, n
= 16 650), and 12.7 ± 1.5 μm and 1.09 ± 0.49 × 10
(HeLa, n
= 26 246). This platform could be further adopted to measure numbers of various cytosolic proteins, providing key insights in proteomics at the single-cell level.
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