Myoglobin and C-reactive protein are efficient and reliable early predictors of COVID-19 associated mortality.

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.

Rhomboidal Pt(II) Metallacycle-Based Hybrid Viral Nanoparticles for Cell Imaging.

Supramolecular coordination complexes (SCCs) have emerged as anticancer agents. Tracking the movement of these metallic anticancer agents plays an important role in the field of biomedicines. Herein, we describe a method for tracking the movement of a rhomboidal Pt(II) metallacycle agent using the quantum dots encapsidation in vitro self-assembly system of viral proteins. When incubated with living Vero cells, self-assembly of hybrid viral nanoparticles were employed for simultaneous cell imaging and visual transmission of the Pt(II) metallacycle agent. Considering these results, we believe that the multifunctional biomaterials consisting of a supramolecular coordination complex and quantum dots provide a new alternative for probing of the delivery of Pt(II) metallacycle drugs.

Detectable Serum Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load (RNAemia) Is Closely Correlated With Drastically Elevated Interleukin 6 Level in Critically Ill Patients With Coronavirus Disease 2019.

BACKGROUND: Although the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in respiratory specimens has been widely used to diagnose coronavirus disease 2019 (COVID-19), it is undeniable that serum SARS-CoV-2 nucleic acid (RNAemia) could be detected in a fraction of COVID-19 patients. However, it is not clear whether testing for RNAemia is correlated with the occurrence of cytokine storms or with the specific class of patients. METHODS: This study enrolled 48 patients with COVID-19 admitted to the General Hospital of Central Theater Command, People's Liberation Army, a designated hospital in Wuhan, China. The patients were divided into 3 groups according to the Diagnosis and Treatment of New Coronavirus Pneumonia (sixth edition) guidelines issued by the National Health Commission of China. Clinical and laboratory data were collected, and the serum viral load and interleukin 6 (IL-6) level were determined. RESULTS: Analysis of clinical characteristics of 48 cases of COVID-19 showed that RNAemia was diagnosed only in the critically ill group and seemed to reflect the severity of the disease. Furthermore, the level of the inflammatory cytokine IL-6 in critically ill patients increased significantly, almost 10 times that in other patients. More importantly, the extremely high IL-6 level was closely correlated with the detection of RNAemia (R = 0.902). CONCLUSIONS: Detectable serum SARS-CoV-2 RNA (RNAemia) in patients with COVID-19 was associated with elevated IL-6 concentration and poor prognosis. Because elevated IL-6 may be part of a larger cytokine storm that could worsen outcome, IL-6 could be a potential therapeutic target for critically ill patients with an excessive inflammatory response.

Cell Type-Dependent Specificity and Anti-Inflammatory Effects of Charge-Reversible MSNs-COS-CMC for Targeted Drug Delivery in Cervical Carcinoma.

The surface charge of nanocarriers inevitably affects drug delivery efficiency; however, the cancer cell specificity, anti-inflammatory effects, and charge-reversal points remain to be further addressed in biomedical applications. The aim of this study was to comprehensively assess the cancer cell specificity of DOX-loaded mesoporous silica-chitosan oligosaccharide-carboxymethyl chitosan nanoparticles (DOX@MSNs-COS-CMC) in MCF-7 and HeLa cells, inhibit the production of inflammatory cytokines, and improve the drug accumulation in the tumor site. Intracellular results reveal that the retention time prolonged to 48 h in both HeLa and MCF-7 cells at pH 7.4. However, DOX@MSNs-COS-CMC exhibited a cell type-dependent cytotoxicity and enhanced intracellular uptake in HeLa cells at pH 6.5, due to the clathrin-mediated endocytosis and macropinocytosis in HeLa cells in comparison with the vesicular transport in MCF-7 cells. Moreover, Pearson's correlation coefficient value significantly decreased to 0.25 after 8 h, prompting endosomal escape and drug delivery into the HeLa nucleus. After the treatment of MSNs-COS-CMC at 200 µg/mL, the inflammatory cytokines IL-6 and TNF-α level decreased by 70% and 80%, respectively. Tumor inhibition of DOX@MSNs-COS-CMC was 0.4 times higher than free DOX, alleviating cardiotoxicity and inflammation in the HeLa xenograft tumor model. Charge-reversible DOX@MSNs-COS-CMC could be a possible candidate for clinical therapy of cervical carcinoma.

Design and biosynthesis of functional protein nanostructures.

Proteins are one of the major classes of biomolecules that execute biological functions for maintenance of life. Various kinds of nanostructures self-assembled from proteins have been created in nature over millions of years of evolution, including protein nanowires, layers and nanocages. These protein nanostructures can be reconstructed and equipped with desired new functions. Learning from and manipulating the self-assembly of protein nanostructures not only help to deepen our understanding of the nature of life but also offer new routes to fabricate novel nanomaterials for diverse applications. This review summarizes the recent research progress in this field, focusing on the characteristics, functionalization strategies, and applications of protein nanostructures.

Development of microfluidic platform capable of high-throughput absolute quantification of single-cell multiple intracellular proteins from tumor cell lines and patient tumor samples.

Quantification of single-cell proteins plays key roles in cell heterogeneity while due to technical limitations absolute numbers of multiple intracellular proteins from large populations of single cells were still missing, leading to compromised results in cell-type classifications. This paper presents a microfluidic platform capable of high-throughput absolute quantification of single-cell multiple types of intracellular proteins where cells stained with fluorescent labelled antibodies are aspirated into the constriction microchannels with excited fluorescent signals detected and translated into numbers of binding sites of targeted proteins based on calibration curves formed by flushing gradient solutions of fluorescent labelled antibodies directly into constriction microchannels. Based on this approach, single-cell numbers of binding sites of ß-actin, α-tubulin and ß-tubulin from tens of thousands of five representative tumor cell lines were first quantified, reporting cell-type classification rates of 83.0 ± 7.1%. Then single-cell numbers of binding sites of ß-actin, biotin and RhoA from thousands of five tumor cell lines with varieties in malignant levels were quantified, reporting cell-type classification rates of 93.7 ± 2.8%. Furthermore, single-cell numbers of binding sites of Ras, c-Myc and p53 from thousands of cells derived from two oral tumor lines of CAL 27, WSU-HN6 and two oral tumor patient samples were quantified, contributing to high classifications of both tumor cell lines (98.6%) and tumor patient samples (83.4%). In conclusion, the developed microfluidic platform was capable of quantifying multiple intracellular proteins from large populations of single cells, and the collected data of protein expressions enabled effective cell-type classifications.

Quantitative profiling of integrin αvß3 on single cells with quantum dot labeling to reveal the phenotypic heterogeneity of glioblastoma.

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 αvß3, on glioblastoma cells at the single cell level. The results revealed that integrin αvß3 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 µm2 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 αvß3 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.

A Microfluidic Fluorescent Flow Cytometry Capable of Quantifying Cell Sizes and Numbers of Specific Cytosolic Proteins.

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 ß-actins at the single-cell level were quantified as 14.2 ± 1.7 µm and 9.62 ± 4.29 × 105 (A549, ncell = 14 242), 13.0 ± 2.0 µm and 6.46 ± 3.34 × 105 (Hep G2, ncell = 35 932), 13.8 ± 1.9 µm and 1.58 ± 0.90 × 106 (MCF 10 A, ncell = 16 650), and 12.7 ± 1.5 µm and 1.09 ± 0.49 × 106 (HeLa, ncell = 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.

[Synthesis of multi-dimensional nano biostructures and biodevices through self-assembly of biomacromolecules].

There are various nanostructures in biological system. They are made of biological molecules via self-assembly with well-organized architectures and specific functions. These nanostructures can be grouped into lines, layers and cages, corresponding to one-, two- and three-dimensional structures, respectively. The bionanostructures can serve as the models or templates for biosynthesis of biodevices with desired functions by rational design. Proof-of-concept studies have shown their attractive performance in practical applications, e.g., biosensors, catalysis, tumor thermo-therapy, drug delivery, tissue engineering, and batteries.

A microfluidic flow cytometer enabling absolute quantification of single-cell intracellular proteins.

Quantification of single-cell proteomics provides key insights into cellular heterogeneity while conventional flow cytometry cannot provide absolute quantification of intracellular proteins of single cells due to the lack of calibration approaches. This paper presents a constriction channel (with a cross sectional area smaller than cells) based microfluidic flow cytometer, capable of collecting copy numbers of specific intracellular proteins. In this platform, single cells stained with fluorescence labelled antibodies were forced to squeeze through the constriction channel with the fluorescence intensities quantified and since cells fully filled the constriction channel during the squeezing process, solutions with fluorescence labelled antibodies were flushed into the constriction channel to obtain calibration curves. By combining raw fluorescence data and calibration curves, absolute quantification of intracellular proteins was realized. As a demonstration, copy numbers of beta-actin of single tumour cells were quantified to be 0.90 ± 0.30 µM (A549, ncell = 14 228), 2.34 ± 0.70 µM (MCF 10A, ncell = 2455), and 0.98 ± 0.65 µM (Hep G2, ncell = 6945). The travelling time for individual cells was quantified to be roughly 10 ms and thus a throughput of 100 cells per s can be achieved. This microfluidic system can be used to quantify the copy numbers of intracellular proteins in a high-throughput manner, which may function as an enabling technique in the field of single-cell proteomics.

Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. FROM THE CLINICAL EDITOR: Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications.

Quantum dot-induced viral capsid assembling in dissociation buffer.

Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.

Construction of bifunctional phage display for biological analysis and immunoassay.

A phage display-based bifunctional display system was developed for simple and sensitive immunoassay. The resulting bifunctional phage could simultaneously display a few single-chain variable fragment (ScFv) and many copies of the gold-binding peptide on its surface, thereby mediating antigen recognition and signal amplification. As a demonstration study, it was possible for bifunctional phage-based immunoassay to identify Bacillus anthracis spores from other Bacillus strains with detection sensitivity 10-fold higher than that of conventional phage enzyme-linked immunosorbent assay (ELISA). This protocol may be applied to build other bifunctional phage clones for broad applications (e.g., immunoassay kits, affinity biosensors, biorecognition assays).