Determination of 23 related analytes in bone marrow fluid and hippocampus of C57BL/6 mice following cerebral ischemia-reperfusion injury by HPLC-FLD.

The study of brain diseases has long been of interest to researchers worldwide, and stroke is the third leading cause of death that threatens human health. At the same time, cerebral ischemia-reperfusion injury is closely associated with high rates of disability and mortality. The conditions of the 6-aminoquinolyl N-hydroxysccinimidyl carbamate method for the derivatization of amino acids in the bone marrow fluid and hippocampus of C57BL/6 mice with cerebral ischemia-reperfusion injury were explored and optimized, such as the column temperature, concentration of derivatization reagents and mobile phase concentration. The mobile phase consisted of 20 mm sodium acetate solution (phosphoric acid to adjust pH 5.0) and 60% acetonitrile solution at a flow rate of 1 ml min-1 . The 23 analytes were separated and determined in a gradient elution procedure; the correlation coefficient r was >0.9990 in the range 0.1-8.0 µg ml-1 . The results showed that the content of relevant analytes was significantly changed in the cerebral ischemia-reperfusion injury model, and the method was suitable for the simultaneous determination of 23 amino acids in the bone marrow fluid and hippocampus of C57BL/6 mice.

Heat-Denatured Lysozyme is a Novel Potential Non-alcoholic Disinfectant Against Respiratory Virus.

Respiratory diseases are significant recurrent threats to global public health. Since the 1918 Spanish flu pandemic, seasonal influenza viruses continue to cause epidemics around the world each year. More recently, the COVID-19 global pandemic conducted a public health crisis with more than 6 million deaths and it also severely affected the global economy. Due to the phenomenon that people get infection from objects carrying viruses, it has aroused people's attention to home disinfection. As there is no ideal existing common domestic disinfectant, new and safer antiviral disinfectants are urgently needed. Lysozyme is a natural antibacterial agent widespread in nature and widely used in healthcare and food industry because of is recognized safety. Recently, it has been shown that thermally denatured lysozyme has the ability to kill murine norovirus and hepatitis A virus. In our study, we also demonstrated that heat-denatured lysozyme (HDLz) had an antiviral effect against H1N1 influenza A virus, and we optimized its antiviral activities by testing different heating denaturation conditions, to generalize this property, using pseudotype virus neutralization assay, we found that HDLz can also inhibit the entry of H5N1, H5N6, and H7N1 avian influenza viruses as well as SARS-CoV and SARS-CoV-2 particles in cell with IC50 at the ng/mL range. Finally, using western blot analysis, we provide evidence that HDLz polymerization correlates with antiviral effect, which may be a precious possible quality control test. Altogether, our data support HDLz as a powerful anti-respiratory virus disinfectant as a sole or additive of current disinfectants to reduce concentration of toxic component.

A renewable electrochemical sensor based on a self-assembled framework of chiral molecules for efficient identification of tryptophan isomers.

Molecular self-assembly provides a reasonably effective strategy for the design and construction of chiral sensors. Here, Cu2+ was connected to ß-cyclodextrin (ß-CD) through coordination to synthesize Cu2-ß-CD, subsequently assembled with ammoniated chitosan-MWCNTs (NH2-CS-MWCNTs) by the effect of coordination driver to form a chiral sensing interface Cu2-ß-CD/NH2-CS-MWCNTs. Using the electrochemical method, the valid recognition of tryptophan (Trp) isomers was achieved on the self-assembly interface. Under the optimal experimental conditions, the developed sensor exhibited good linearity and satisfactorily renewable ability. Cu2-ß-CD/NH2-CS-MWCNTs/GCE showed the capacity to predict the ratio of D-Trp and L-Trp in racemic mixtures and the possibility of qualitative and quantitative determination for Trp isomers. Finally, the electrochemical sensor was used to detect the Trp enantiomers in rat serum, further verifying the feasibility of the sensor in the determination of actual samples. Therefore, the electrochemical chiral sensor not only is used for the recognition of Trp enantiomers but shows great potential in practical applications.

Determination of 23 Amino Acids and Amines in Plasma and Cortex of C57BL/6 Mice with HPLC-FLD.

Using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as a pre-column derivatization reagent, optimized derivatization and chromatography parameters, a simple high-performance liquid chromatography fluorescence detector (HPLC-FLD) method was developed and validated to determine 23 related amines in plasma and cortex of C57BL/6 mice with cerebral ischemia-reperfusion injury. The prepared samples were separated on a ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 µm) with 60% acetonitrile (ACN) and 20 mM sodium acetate solution (pH adjusted to 5.0 by phosphoric acid). All analytes achieved good separation within 1.2 h at a flow rate of 1 mL/min. The limits of detection and limits of detection quantitation of the method were ranged from (0.1-9.2) to (0.3-30.6) ng/mL, respectively. The analytical method was apt for simultaneously determining 23 amino acids in plasma and cortex. Our results revealed that the relevant amino acids were significantly altered (P < 0.05) in C57BL/6 mice.

Isolation, characterization, and structure-based engineering of a neutralizing nanobody against SARS-CoV-2.

SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 µg mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.

Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants.

SARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a K D of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC50 of 0.41 µg mL-1. Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC50 values ranging from 0.35 to 1.66 µg mL-1 for the Alpha/Beta/Gamma/Delta and an IC50 of 0.66 µg mL-1 for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a "conformation competition" mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding.

Three Dimensional Printing-Based Strategies for Functional Cartilage Regeneration.

IMPACT STATEMENT: This article primarily reviews the applications of three-dimensional printing in cartilage tissue engineering at different anatomical locations and summarizes their strengths and limitations. In addition, we believe that four-dimensional concept and biological microenvironment should not be ignored for functional cartilage regeneration in the future. Finally, we hope the review provide scientist inspiration with constructing anisotropic tissue-engineered organ or tissue.