Utilizing noncatalytic ACE2 protein mutant as a competitive inhibitor to treat SARS-CoV-2 infection.

Introduction: Angiotensin converting-enzyme 2 (ACE2) is an enzyme catalyzing the conversion of angiotensin 2 into angiotensin 1-7. ACE2 also serves as the receptor of several coronaviruses, including SARS-CoV-1 and SARS-CoV-2. Therefore, ACE2 could be utilized as a therapeutic target for treating these coronaviruses, ideally lacking enzymatic function. Methods: Based on structural analysis, specific mutations were introduced to generate mutants of ACE2 and ACE2-Fc (fusion protein of ACE2 and Fc region of IgG1). The enzyme activity, binding affinity, and neutralization abilities were measured. Results and discussion: As predicted, five mutants (AMI081, AMI082, AMI083, AMI084, AMI090) have completely depleted ACE2 enzymatic activities. More importantly, enzyme-linked receptor-ligand assay (ELRLA) and surface plasmon resonance (SPR) results showed that 2 mutants (AMI082, AMI090) maintained binding activity to the viral spike proteins of SARS-CoV-1 and SARS-CoV-2. In An in vitro neutralization experiment using a pseudovirus, SARS-CoV-2 S1 spike protein-packed lentivirus particles, was also performed, showing that AMI082 and AMI090 significantly reduced GFP transgene expression. Further, in vitro virulent neutralization assays using SARS-CoV-2 (strain name: USA-WA1/2020) showed that AMI082 and AMI090 had remarkable inhibitory effects, indicated by comparable IC50 to wildtype ACE2 (5.33 µg/mL). In addition to the direct administration of mutant proteins, an alternative strategy for treating COVID-19 is through AAV delivery to achieve long-lasting effects. Therefore, AAV5 encoding AMI082 and AMI090 were packaged and transgene expression was assessed. In summary, these ACE2 mutants represent a novel approach to prevent or treat COVID-19 and other viruses with the same spike protein.

Evaluation of recombinant baculovirus clearance during rAAV production in Sf9 cells using a newly developed fluorescent-TCID50 assay.

Introduction: Recombinant adeno-associated virus (rAAV) vectors provide a safe and efficient means for in vivo gene delivery, although its large-scale production remains challenging. Featuring high manufacturing speed, flexible product design, and inherent safety and scalability, the baculovirus/Sf9 cell system offers a practical solution to the production of rAAV vectors in large quantities and high purity. Nonetheless, removal and inactivation of recombinant baculoviruses during downstream purification of rAAV vectors remain critical prior to clinical application. Methods: The present study utilized a newly developed fluorescent-TCID50 (F-TCID50) assay to determine the infectious titer of recombinant baculovirus (rBV) stock after baculovirus removal and inactivation, and to evaluate the impact of various reagents and solutions on rBV infectivity. Results and discussion: The results showed that a combination of sodium lauryl sulfate (SLS) and Triton X-100 lysis, AAVx affinity chromatography, low pH hold (pH3.0), CsCl ultracentrifugation, and NFR filtration led to effective removal and/or inactivation of recombinant baculoviruses, and achieved a log reduction value (LRV) of more than 18.9 for the entire AAV purification process. In summary, this study establishes a standard protocol for downstream baculovirus removal and inactivation and a reliable F-TCID50 assay to detect rBV infectivity, which can be widely applied in AAV manufacturing using the baculovirus system.

Fast Li+ Transport Polyurethane-Based Single-Ion Conducting Polymer Electrolyte with Sulfonamide Side chains in the Hard Segment for Lithium Metal Batteries.

Single-ion conducting polymer electrolytes (SICPEs) are considered as one of the most promising candidates for achieving lithium metal batteries (LMBs). However, the application of traditional SICPEs is hindered by their low ionic conductivity and poor mechanical stability. Herein, a self-standing and flexible polyurethane-based single-ion conductor membrane was prepared via covalent tethering of the trifluoromethanesulfonamide anion to polyurethane, which was synthesized using a facile reaction of diisocyanates with poly(ethylene oxide) and 3,5-diaminobenzoic acid (or 3,5-dihydroxybenzoic acid). The polymer electrolyte exhibited excellent ionic conductivity, mechanical properties, lithium-ion transference number, thermal stability, and a broad electrochemical window because of the bulky anions and unique two-phase structures with lithium-ion nanochannels in the hard domains. Consequently, the plasticized electrolyte membrane showed exceptional stability and reliability in a Li||Li symmetric battery. The assembled LiFePO4||Li battery exhibited an outstanding capacity (∼180 mA h g-1), Coulombic efficiency (>96%), and capacity retention. This research provides a promising polymer electrolyte for high-performance LMBs.

State Evaluation Method of Robot Lubricating Oil Based on Support Vector Regression.

Recently, the development of the Industrial Internet of Things (IIoT) has led enterprises to re-examine the research of the equipment-state-prediction models and intelligent manufacturing applications. Take industrial robots as typical example. Under the effect of scale, robot maintenance decision seriously affects the cost of spare parts and labor deployment. In this paper, an evaluation method is proposed to predict the state of robot lubricating oil based on support vector regression (SVR). It would be the proper model to avoid the structural risks and minimize the effect of small sample volume. IIoT technology is used to collect and store the valuable robot running data. The key features of the running state of the robot are extracted, and the machine learning model is applied according to the measured element contents of the lubricating oil. As a result, the cost of spare parts consumption can be saved for more than two million CNY per year.

Significantly Improved Dielectric Performance of Poly(1-butene)-Based Composite Films via Filling Polydopamine Modified Ba(Zr0.2Ti0.8)O3-Coated Multiwalled Carbon Nanotubes Nanoparticles.

The low dielectric constant of the nonpolar polymer poly(1-butene) (PB-1) limits its application as a diaphragm element in energy storage capacitors. In this work, Ba(Zr0.2Ti0.8)O3-coated multiwalled carbon nanotubes (BZT@MWCNTs) were first prepared by using the sol-gel hydrothermal method and then modified with polydopamine (PDA) via noncovalent polymerization. Finally, PB-1 matrix composite films filled with PDA-modified BZT@MWCNTs nanoparticles were fabricated through a solution-casting method. Results indicated that the PDA-modified BZT@MWCNTs had good dispersion and binding force in the PB-1 matrix. These characteristics improved the dielectric and energy storage performances of the films. Specifically, the PDA-modified 10 vol% BZT@ 0.5 vol% MWCNTs/PB-1 composite film exhibited the best dielectric performance. At 1 kHz, the dielectric constant of this film was 25.43, which was 12.7 times that of pure PB-1 films. Moreover, its dielectric loss was 0.0077. Furthermore, under the weak electric field of 210 MV·m-1, the highest energy density of the PDA-modified 10 vol% BZT@ 0.5 vol% MWCNTs/PB-1 composite film was 4.57 J·cm-3, which was over 3.5 times that of PB-1 film (≈1.3 J·cm-3 at 388 MV·m-1).

Crystallization and Dielectric Properties of MWCNT /Poly(1-Butene) Composite Films by a Solution Casting Method.

In this work, poly(1-butene) (PB-1) composite films with multi-walled carbon nanotubes (MWCNT) were prepared by a solution casting method. The relationship between the dielectric properties and the crystal transformation process of the films was investigated. It was indicated that there were two crystal forms of I and II of PB-1 during the solution crystallization process. With the prolongation of the phase transition time, form II was converted into form I. The addition of the conductive filler (MWCNT) accelerated the rate of phase transformation and changed the nucleation mode of PB-1. The presence of crystal form I in the system increased the breakdown strength and the dielectric constant of the films and reduced the dielectric loss, with better stability. In addition, the dielectric constant and the dielectric loss of the MWCNT/PB-1 composite films increased with the addition of MWCNT, due to the interfacial polarization between MWCNT and PB-1 matrix. When the mass fraction of the MWCNT was 1.0%, the composite film had a dielectric constant of 43.9 at 25 °C and 103 Hz, which was 20 times that of the original film.

A total solid-phase synthesis of DILP8.

We have developed a cysteine anchoring method for the synthesis of DILP8 and its analogues. The first is to synthesis of DILP8A SS13-18, C14-MeOBzl, C24-Acm and activate it as DILP8A S13-18, C14-SSPyr C24-Acm. A next step is to synthesize the DILP8BC16-Acm. The desired peptide, DILP8 with Cys(Acm) at A-24 and B-16, was then dissolved in 75% HOAc by addition of Iodine in MeOH and 4M HCl in dioxane. The reaction mixture was monitored by HPLC and the excess iodine was reduced with ascorbic acid. Purification of the peptide was achieved by HPLC. Pure synthetic DILP8 showed a single peak on analytical HPLC with corrected molecular ion. By using the above methods, enough peptide and highly homogenous pure DLP8 were generated.

The formation mechanism of the beta-TCP phase in synthetic fluorohydroxyapatite with different fluorine contents.

Synthetic hydroxyapatite (HAP) and fluorohydroxyapatite (F(x)AP) products may form the beta-tricalcium phosphate (beta-TCP) phase in a calcination process. The beta-TCP phase has a greater tendency for degradation in vivo than HAP and F(x)AP. Hence, controlling the content of the beta-TCP phase in the apatite is a pivotal factor to affect their lifetime and stability in vivo. It is particularly important to explore the formation mechanism of the beta-TCP phase in synthetic apatite. In this work, F(x)AP products with a chemical composition of Ca(10)(PO(4))(6)(OH)(2-x)F(x) are synthesized, with x = 0, 0.4, 0.8, 1.2, 1.6 and 2.0, using a precipitation method and a calcination process. The effect of fluorine substitution for hydroxyl is investigated by using x-ray diffraction analysis, Fourier transform infrared spectroscopy, and thermogravimetry and differential thermal analysis. The results show that addition of fluorine forms F(x)AP that exhibits high thermal stability. The beta-TCP phase produced as a result of the structural refinement by heat treatment is gradually reduced and dramatically suppressed with the fluorine content.

Prevalence and risk factors of fatty liver disease in the Shuiguohu district of Wuhan city, central China.

BACKGROUND: Fatty liver disease (FLD) is highly prevalent in Western countries, but recent data have shown that FLD is also emerging in China. AIM: To investigate the prevalence and risk factors of FLD in the Shuiguohu district of Wuhan city, central China, during 1995-2004. METHODS: 12247 individuals (7179 men and 5068 women) over 18 years of age who were living in the area were investigated for FLD in the Zhongnan Hospital of Wuhan University from 1995 to 2004. FLD was determined by the ultrasonographic method. Height, weight, blood pressure, fasting blood sugar, alanine aminotransferase, total cholesterol and triglyceride were determined by routine laboratory methods. RESULTS: The prevalence of FLD was 12.5% in 1995, and rose gradually to 24.5% by 2003-4. The prevalence was twice as high in men (28.1%) as in women (13.8%), and increased with age in females, and males <60 years of age. Multivariate analysis showed that several risk factors were profoundly associated with the prevalence of FLD, including male sex, old age, obesity, hyperlipidaemia (cholesterol or triglyceride), fasting hyperglycemia and hypertension. CONCLUSION: The prevalence of FLD in the Shuiguohu district of Wuhan city, central China, was shown to have increased during the 10-year period, 1995 to 2004. The FLD was found to be closely associated with sex, age, obesity and other metabolic syndrome features.

Metastin-like immunoreactivity in the rat medulla oblongata and spinal cord.

Metastin, the product of metastasis suppressor gene KiSS-1, is proposed to be the natural ligand for the G-protein-coupled receptor GPR54, known also as AXOR12. This immunohistochemical study, using a rabbit polyclonal antiserum against the human metastin fragment (45-54)-NH(2), showed that in rats metastin-like immunoreactivity (MTS-LI) was present in neurons of the nucleus of the solitary tract and caudoventrolateral reticular nucleus, and in cell processes of the spinal trigeminal tract and lateral reticular nucleus. MTS-LI was confined mainly to neurons and fibers at or caudal to the area postrema. In the spinal cord, MTS-LI cell processes formed a dense plexus in superficial layers I and II of the dorsal horn. The pattern of distribution of MTS-LI in the medulla and spinal cord suggests that this novel peptide may participate in autonomic and sensory neural signaling.

Photocatalytic decomposition of toluene by TiO2 film as photocatalyst.

This paper reports on the degradation of gaseous toluene by the photocatalytic oxidation process. The photocatalyst, TiO2 film, was prepared by the sol-gel method and was coated on porous nickel. The photocatalytic decomposition of toluene was carried out in a recirculating batch reactor. The factors that affect toluene decomposition were studied, including particle size of the catalyst, initial toluene concentration, flow rate of toluene in the reactor, relative humidity and reactive temperature. In addition, deactivation of the catalyst is also discussed. The experiment results showed that toluene can be decomposed effectively by the photocatalytic oxidation process, and that toluene degradation obeys the first-order reaction.

Characterization of RhoA-binding kinase ROKalpha implication of the pleckstrin homology domain in ROKalpha function using region-specific antibodies.

Rho-binding kinase alpha (ROKalpha) is a serine/threonine kinase with multiple functional domains involved in actomyosin assembly. It has previously been documented that the C terminus part of ROKalpha interacts with the N-terminal kinase domain and thereby regulates its catalytic activity. Here we used antibodies against different domains of ROKalpha and were able to reveal some structural aspects that are essential for the specific functions of ROKalpha. Antibodies against the kinase domain revealed that this part of the protein is highly complex and inaccessible. Further experiments confirmed that this domain could undergo inter- and intramolecular interactions in a complex manner, which regulates the kinase catalytic activity. Other antibodies that raised against the coiled-coil domain, Rho binding domain, and the pleckstrin homology (PH) domain were all effective in recognizing the native proteins in an immunoprecipitation assay. Only the anti-Rho binding domain antibodies could activate the kinase independent of RhoA. The PH antibodies had no apparent effects on the catalytic activity but were effective in blocking actomyosin assembly and cell contractility. Likewise, mutations of the PH domains can abrogate its dominant negative effects on actin morphology. The subsequent disruption of endogenous ROK localization to the actomyosin network by overexpressing the PH domain is supportive of a role of the PH domain of ROK in targeting the kinase to these structures.