Nuclear localization signal peptides enhance genetic transformation of Dunaliella salina.

BACKGROUND: Dunaliella salina (D. salina) expression system shows a very attractive application prospect, but it currently has a technical bottleneck, namely the low or unstable expression of recombinant proteins. Given the characteristics of cell-penetrating peptides or/and nuclear localization signal (NLS) peptides, this study is the first attempt to improve the transformation rate of foreign gene with trans-activating transcriptional (TAT) protein or/and NLS peptides. METHODS AND RESULTS: Using salt gradient method, exogenous plasmids were transferred into D. salina cells with TAT or TAT/NLS complexes simultaneously. The ß-glucuronidase gene expression was identified by means of histochemical stain and RT-qPCR detection. Through observation with light microscope, TAT-mediating cells exhibit an apparent cytotoxicity even at ratios of 0.5, no significant toxicity was noted in the TAT/plasmid/NLS complex group. It is obvious that with the addition of peptides the toxicity decreases significantly. Histochemical staining showed that the transformants presented blue color under light microscope, but the negative control and blank control are not. Furthermore, based on a TAT/plasmids ratio of 4 with 10 µg NLS peptides mediation, RT-qPCR results demonstrated that the transcripts of target gene were increased by 269 times than that of control group. CONCLUSIONS: This study demonstrated that combination of TAT and NLS peptides can significantly improve the transformation rate and expression level of foreign gene in D. salina system. It offers a promising way for promoting the application and development of D. salina bioreactor.

Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells.

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

The ultrasonically treated nanoliposomes containing PCV2 DNA vaccine expressing gC1qR binding site mutant Cap is efficient in mice.

Nowadays, vaccines are broadly used to prevent porcine circovirus type 2 (PCV2) infection-induced expenditures, but the virus is still spreading among pigs. The current PCV2 vaccines all rely on the immunogenicity of Cap, yet our previous studies found that Cap is also the major component mediating the PCV2 infection-induced immune suppression through its interaction with host gC1qR. Thereby, new vaccines are still necessary for PCV2 prevention and control. In this study, we constructed a new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap. We introduced the Intron A and WPRE elements into the vector to improve the Cap expression level, and fused the IL-2 secretory signal peptides to the N-terminal of Cap to mediate the secretion of Cap. We also screened and selected chemokines CXCL12, CCL22, and CCL25 to migrate dendritic cells. In addition, we contained the vectors with PEI and then ultrasonic them into nano size to enhance the entrance of the vectors. Finally, the animal experiments showed that the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap could induce stronger humoral and cellular immune responses than the PCV2 DNA vaccine expressing the wild-type Cap and the non-ultrasonic treated PCV2 DNA vaccine in mice, and protect the mice from PCV2 infection and lung lesions. The results indicate the new PCV2 DNA vaccine expressing the gC1qR binding site mutant Cap has a certain development value, and provide new insight into the development of novel PCV2 vaccines.

TIST: Transcriptome and Histopathological Image Integrative Analysis for Spatial Transcriptomics.

Sequencing-based spatial transcriptomics (ST) is an emerging technology to study in situ gene expression patterns at the whole-genome scale. Currently, ST data analysis is still complicated by high technical noises and low resolution. In addition to the transcriptomic data, matched histopathological images are usually generated for the same tissue sample along the ST experiment. The matched high-resolution histopathological images provide complementary cellular phenotypical information, providing an opportunity to mitigate the noises in ST data. We present a novel ST data analysis method called transcriptome and histopathological image integrative analysis for ST (TIST), which enables the identification of spatial clusters (SCs) and the enhancement of spatial gene expression patterns by integrative analysis of matched transcriptomic data and images. TIST devises a histopathological feature extraction method based on Markov random field (MRF) to learn the cellular features from histopathological images, and integrates them with the transcriptomic data and location information as a network, termed TIST-net. Based on TIST-net, SCs are identified by a random walk-based strategy, and gene expression patterns are enhanced by neighborhood smoothing. We benchmark TIST on both simulated datasets and 32 real samples against several state-of-the-art methods. Results show that TIST is robust to technical noises on multiple analysis tasks for sequencing-based ST data and can find interesting microstructures in different biological scenarios. TIST is available at http://lifeome.net/software/tist/ and https://ngdc.cncb.ac.cn/biocode/tools/BT007317.