CRISPR-Cas9: A method for establishing rat models of drug metabolism and pharmacokinetics.

The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9, a super-selective and precise gene editing tool. CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell, and presents great potential in disease treatment and animal model construction. In recent years, CRISPR-Cas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics (DMPK), such as Cyp, Abcb1, Oatp1b2 gene knockout rats. These new rat models are not only widely used in the study of drug metabolism, chemical toxicity, and carcinogenicity, but also promote the study of DMPK related mechanism, and further strengthen the relationship between drug metabolism and pharmacology/toxicology. This review systematically introduces the advantages and disadvantages of CRISPR-Cas9, summarizes the methods of establishing DMPK rat models, discusses the main challenges in this field, and proposes strategies to overcome these problems.

Investigation of the content differences of arachidonic acid metabolites in a mouse model of breast cancer by using LC-MS/MS.

Arachidonic acid (AA) is closely associated with breast cancer. In addition to the two metabolic pathways regulated by cyclooxygenase and lipoxygenase, AA has a third metabolic pathway through which cytochrome P450 (CYP) enzymes produce hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs). The targeted CYP-mediated pathway of AA can not only kill cancer cells but also inhibit the interstitial microenvironment around a tumor. Therefore, it makes sense to identify potential biomarkers from the AA metabolome for the diagnosis and treatment of breast cancer. This study established a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of AA and its main metabolites, EETs and HETEs, in MMTV-PyMT mice, a spontaneous breast cancer mouse model. The results showed that there were significant differences in the concentrations of AA, 12-HETE, 19-HETE and 8,9-EET in plasma and tumor tissues between normal and MMTV-PyMT mice. Therefore, the eicosanoids mentioned above may be used as new biomarkers for breast cancer diagnosis. This study provides a new perspective for the recognition and diagnosis of breast cancer.

A single-cell identification and capture chip for automatically and rapidly determining hydraulic permeability of cells.

The hydraulic permeability of the lipid bilayer membrane of a single cell, a very important parameter in biological and medical fields, has been attracting increasing attention. To date, methods developed to determine this permeability are either operation-complicated or time-consuming. Therefore, we developed a chip for automatically and rapidly determining the permeability of cells that integrates microfluidics and cell impedance analysis. The chip is designed to automatically identify a single cell, capture the cell, and record the volume change in that cell. We confirmed the abilities of single-cell identification and capture with the upper and lower voltage thresholds determined, validated the performance of the differential electrode design for accurate cell volume measurements, deduced the extracellular osmotic pressure change in the presence of a hypertonic solution according to fluorescence intensity, and demonstrated the single-cell volume change recorded by the chip. Then, the accuracy of the permeability determined with the chip was verified using HeLa cells. Finally, the permeability of human-induced pluripotent stem cells (hiPSCs) was determined to be 0.47 ± 0.03 µm/atm/min. Using the chip, the permeability can be determined within 5 min. This study provides insights for the new design of an automatic single-cell identification and capture chip for single cell-related studies. Graphical abstract.

Effect of High Pressure Treatment on Interfacial Properties, Structure and Oxidative Stability of Soy Protein Isolate-Stabilized Emulsions.

The effect of high pressure homogenization (HPH) treatment (40 to 200MPa) on the structure, interfacial properties and oxidative stability of soy protein isolate emulsions were investigated. After HPH process, emulsifying activity (EAI) and emulsion stability (ESI) could be improved by 114% and 125%, percentage of protein adsorbed to oil-in-water emulsions significantly improved when the pressure was not more than 160 MPa. SDS-PAGE showed that the HPH caused more low molecular mass subunits as the samples were treated below 160 MPa. FTIR traced the changes of four conformations in the secondary structure. Within a suitable pressure range, the apparent viscosity increased with the increasing of pressure, which reflected shear thinning; rheological measurement of emulsions showed G'>G'', G' and G'' both increased significantly with frequency increasing; emulsions treated by HPH had better oxidative stability. This paper suggested the HPH was a useful method for preparation the highly viscous and stable emulsions.

Effects of homogenization on the molecular flexibility and emulsifying properties of soy protein isolate.

The sensitivity of soy protein isolate (SPI) to trypsin was characterized by its flexibility. The effects of different homogenization conditions on soy protein isolate flexibility and emulsifying properties were investigated. Set the homogenization pressure was 120 MPa (megapascal) and the homogenous number of times is 0-4 times, the flexibility increases with the increase of the homogenization times (0-3 times), the change trend of flexibility is not obvious (3-4 times). When the homogenization times was 0-3 times, the emulsifying activity increases, and the emulsifying activity was the strongest at 3 times, after homogenization 3 times, the change trend of emulsifying activity is not obvious, the trend of emulsification stability and emulsification activity were similar. The surface hydrophobicity increases with the increase of homogenization times, while the turbidity decreases. The other structural indicators such as Ultraviolet scanning and endogenous tryptophan fluorescence spectroscopy suggest that the structure of SPI becomes more stretch as the flexibility increases.

Clinical application of antenatal genetic diagnosis of osteogenesis imperfecta type IV.

BACKGROUND: Clinical analysis and genetic testing of a family with osteogenesis imperfecta type IV were conducted, aiming to discuss antenatal genetic diagnosis of osteogenesis imperfecta type IV. MATERIAL/METHODS: Preliminary genotyping was performed based on clinical characteristics of the family members and then high-throughput sequencing was applied to rapidly and accurately detect the changes in candidate genes. RESULTS: Genetic testing of the III5 fetus and other family members revealed missense mutation in c.2746G>A, pGly916Arg in COL1A2 gene coding region and missense and synonymous mutation in COL1A1 gene coding region. CONCLUSIONS: Application of antenatal genetic diagnosis provides fast and accurate genetic counseling and eugenics suggestions for patients with osteogenesis imperfecta type IV and their families.