Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system.

BACKGROUND: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips. METHODS: We firstly delivered a 13 kb Cas9-EGFP and a 3.5 kb pMAX-GFP vector into PFFs via 7 programs recommended by the Lonza basic protocol. We then tested 6 customized dual-electroporation programs for delivering the 13 kb plasmid into PFFs. In addition, we screened potential alternative electroporation buffers to the Nucleofector™ P3 solution. Finally, three CRISPR/Cas9-sgRNAs targeting Rosa26, H11, and Cep112 loci were delivered into PFFs with different single and dual-electroporation programs. RESULTS: Notably lower transfection efficiencies were observed when delivering the 13 kb vector than delivering the 3.5 kb vector in PFFs via the single-electroporation programs. The customized dual-electroporation program FF-113 + CA-137 exhibited higher transfection efficiencies than any of the single-electroporation programs using vessels (98.1%) or strips (89.1%) with acceptable survival rates for the 13 kb vector. Entranster-E buffer generated similar transfection efficiencies and 24-hour survival rates to those from the P3 solution, thus can be used as an alternative electroporation buffer. In the genome-editing experiments, the FF-113 + CA-137 and CA-137 + CA-137 programs showed significantly superior (P < 0.01) efficiencies to ones from the single-electroporation programs in vessels and strips. Entranster-E buffer produced higher indel efficiencies than the P3 buffer. CONCLUSIONS: We markedly increased the delivery efficiencies for a large vector via customized dual-electroporation programs using Lonza 4D-Nucleofector™ system, and Entranster-E buffer can be used as an alternative electroporation buffer to Nucleofector™ P3 buffer.

Comparison of the Value of Color Doppler Ultrasound and Multislice Spiral CT in the Differential Diagnosis of Benign and Malignant Nodules in the Liver.

OBJECTIVE: This study aimed to explore the value of color Doppler ultrasound and multislice spiral CT (MSCT) in the differential diagnosis of benign and malignant nodules in the liver. METHODS: The clinical imaging data of 102 patients with nodular hepatocellular carcinoma (hepatocellular carcinoma group) and 50 patients with focal nodular hyperplasia (FNH) of the liver (FNH group) admitted to our hospital were collected, and their color Doppler ultrasound and MSCT imaging features were retrospectively analyzed to explore the value of their clinical application in the differential diagnosis of benign and malignant nodules in the liver. RESULTS: The sensitivity, accuracy, and negative predictive value of MSCT in the diagnosis of nodular liver cancer were 94.12%, 92.76%, and 88.24%, respectively, which were significantly higher than those of color Doppler ultrasound 79.41%, 84.21%, and 69.12%, and the difference was statistically significant (P < 0.05). CONCLUSION: In conclusion, the value of MSCT in the differential diagnosis of benign and malignant liver nodules was significantly better than color Doppler ultrasound.

Nitrogen-doped carbon dots for sensitive detection of ferric ions and monohydrogen phosphate by the naked eye and imaging in living cells.

Nitrogen doped carbon dots (N-CDs) have been prepared via a one-pot hydrothermal method by using formamide and o-phenylenediamine as the carbon precursors. The as-fabricated N-CDs display excellent water dispersibility, good biocompatibility and anti-photobleaching properties. A strong emission band with an emission maximum (λ fl max) of 556 nm is observed under 450 nm excitation, and a large Stokes shift of 106 nm is presented. However, the fluorescence is quenched by the addition of Fe3+; a good linearity is shown in the range of 0-65 µM with a detection limit as low as 0.85 µM. Fortunately, the quenched fluorescence could be recovered rapidly by the addition of monohydrogen phosphate (HPO4 2-) due to the formation of the stable [N-CDs-Fe3+-HPO4 2-] complex, and a good linearity is exhibited in the range of 0-60 µM with a low detection limit of 0.80 µM for HPO4 2-. A novel "on-off-on" fluorescence response is seen with an obvious color change from yellow-crimson-yellow by the naked eye. In addition, the confocal microscopy images suggest that the as-synthesized N-CDs could serve as a sensitive nanosensor for Fe3+ and HPO4 2- detection, implying the diverse potential application of N-CDs in the biomedical field.

A spiropyran functionalized fluorescent probe for mitochondria targeting and imaging of endogenous hydrogen sulfide in living cells.

A turn-on spiropyran functionalized fluorescein derivative (FMC) is developed for targeting HS- in mitochondria. FMC exhibits very weak fluorescence at 525 nm under the excitation of 470 nm in aqueous solution due to its colorless spiropyran form; upon addition of HS-, a strong fluorescence enhancement by 6.4-fold is observed with spirocycle-opened merocyanine form and rapid trapping kinetics for HS-. FMC has good biocompatibility and high selectivity towards HS- with a detection limit of 88.2 nM and is very sensitive among the reported H2S fluorescent probes. Moreover, the significant colocalization of FMC with Mito Tracker® Deep Red FM in human laryngeal epidermoid carcinoma (HEp-2) cells and the Pearson correlation coefficient of 0.87 together demonstrate that FMC can target and image the endogenous H2S in the mitochondria of living cells.

Clinical value of TV-CDS combined with serum tumor markers in diagnosis of ovarian cancer.

Clinical application value was investigated of transvaginal color Doppler ultrasound (TV-CDS) combined with serum tumor markers carbohydrate antigen 125 (CA125), vascular endothelial growth factor (VEGF) and osteopontin (OPN) in the diagnosis of ovarian cancer (OC). One hundred and six patients with OC [malignant tumor group (MTG)] and fifty patients with benign ovarian diseases [benign control group (BCG)] were selected. Both groups of patients underwent TV-CDS examination. The lesion morphology and internal structure were observed, and the tumor blood flow signal, resistance index (RI) and pulsability index (PI) under ultrasound were determined. Serum CA125 was detected by electrochemiluminescence, and VEGF and OPN levels were detected by enzyme-linked immunosorbent assay. The incidence of irregular lesion morphology, unclear boundary, uneven internal echo, microcalcification and side-acoustic images in OC group (OCG) was significantly higher than that in BCG (P<0.01). As for blood flow grading, most patients in the MTG were in grade II and III, while most patients in the BCG were in grade 0. Compared with BCG, the flow RI and PI in the OCG were significantly reduced (P<0.01). The levels of serum CA125, VEGF and OPN in OCG were significantly higher than those in BCG. The expression levels of serum CA125, VEGF and OPN in OC patients with clinical high stage (stage III and IV), poorly differentiated, ascites, recurrence and metastasis were significantly higher than those in patients with clinical low stage (stage I and II), well differentiated, no ascites and no recurrence and metastasis (P<0.05). With the disappearance of the tumor or the decrease of tumor load, the serum marker levels after treatment were significantly lower than that before treatment (P<0.05). The sensitivity and accuracy of the combined examination in the diagnosis of OC were obviously improved compared with the single and partial combined examinations (P<0.05). In conclusion, combined examination can significantly improve the sensitivity and accuracy of OC, which is conducive to early diagnosis and clinical intervention of OC.