Production of MSTN knockout porcine cells using adenine base-editing-mediated exon skipping.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5' end splice donor and 3' end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5'-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5'-GC) in the conserved sequence (5'-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.

Autophagy and Apoptosis in Acute Brain Injuries: From Mechanism to Treatment.

Significance: Autophagy and apoptosis are two important cellular mechanisms behind brain injuries, which are severe clinical situations with increasing incidences worldwide. To search for more and better treatments for brain injuries, it is essential to deepen the understanding of autophagy, apoptosis, and their interactions in brain injuries. This article first analyzes how autophagy and apoptosis participate in the pathogenetic processes of brain injuries respectively and mutually, then summarizes some promising treatments targeting autophagy and apoptosis to show the potential clinical applications in personalized medicine and precision medicine in the future. Recent Advances: Most current studies suggest that apoptosis is detrimental to brain recovery. Several studies indicate that autophagy can cause unnecessary death of neurons after brain injuries, while others show that autophagy is beneficial for acute brain injuries (ABIs) by facilitating the removal of damaged proteins and organelles. Whether autophagy is beneficial or detrimental in ABIs depends on many factors, and the results from different research groups are diverse or even controversial, making this topic more appealing to be explored further. Critical Issues: Neuronal autophagy and apoptosis are two primary pathological processes in ABIs. How they interact with each other and how their regulations affect the outcome and prognosis of brain injuries remain uncertain, making these answers more critical. Future Directions: Insights into the interplay between autophagy and apoptosis and the accurate regulations of their balance in ABIs may promote personalized and precise treatments in the field of brain injuries. Antioxid. Redox Signal. 38, 234-257.

Trefoil factor 3 promotes pancreatic carcinoma progression via WNT pathway activation mediated by enhanced WNT ligand expression.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related mortality with a dismal prognosis that has changed little over the past few decades. Further understanding of the molecular pathology of PDAC progression is urgently required in order to improve the prognosis of patients with PDAC. Herein, it was observed that trefoil factor 3 (TFF3) expression was elevated in PDAC, and was positively correlated with a worse overall patient survival outcome. Forced expression of TFF3 promoted oncogenic functions of PDAC cells in vitro including cell proliferation, survival, foci formation, cancer stem cell-like behavior and invasion, ex vivo colony growth in 3D-Matrigel, and xenograft growth in vivo. Depletion or pharmacological inhibition of TFF3 inhibited these same processes. RNA-Seq analysis and subsequent mechanistic analyses demonstrated that TFF3 increased the expression of various WNT ligands to mediate WNT pathway activation required for TFF3-stimulated PDAC progression. Combined pharmacological inhibition of TFF3 and WNT signaling significantly attenuated PDAC xenograft growth and potentiated the therapeutic efficacy of gemcitabine in both ex vivo and in vivo models. Hence, a mechanistic basis for combined inhibition of pathways enhancing PDAC progression is provided and suggests that inhibition of TFF3 may assist to ameliorate outcomes in PDAC.

Efficient Oral Delivery of Poorly Water-Soluble Drugs Using Carnitine/Organic Cation Transporter 2-Mediated Polymeric Micelles.

The intestine epithelium is considered to be the most critical obstacle for nanoparticles for oral delivery of water-insoluble and poorly absorbed drugs. Based on the specific transporters located on the apical membrane of the intestinal epithelium, the carnitine-conjugated polymeric micelles targeting to the carnitine/organic cation transporter 2 (OCTN2) were developed by combining carnitine-conjugated poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) with monomethoxy poly(ethylene-glycol)-poly(d,l-lactide). The carnitine-conjugated micelles with favorable stability in gastrointestinal fluid were validated to remarkably increase the cellular internalization and transcellular transport, while these were not the cases in the presence of free carnitine. These were further confirmed by more distribution of the micelles within epithelial cells, on the apical and basolateral side of the epithelium in mice. Additionally, identification of the carnitine-conjugated micelles by OCTN2 was detected to facilitate cellular uptake of the micelles via fluorescence immunoassay. Both clathrin and caveolae/lipid rafts pathways mediated endocytosis and transcellular transport of the carnitine-conjugated micelles, implying the enrichment of endocytic and transcellular transport pathway compared with that of carnitine-unconjugated micelles. Further, the intracellular trafficking process of the carnitine-conjugated micelles was tracked under confocal laser scanning microscopy, which involved in intracellular compartments such as late endosomes, lysosomes, endoplasmic reticulum, and Golgi apparatus as well. In conclusion, the current study provided an efficient strategy to facilitate the oral absorption of water-insoluble and poorly absorbed agents using intestinal transporter-mediated polymeric micelles.

Synergistically Enhanced Antimetastasis Effects by Honokiol-Loaded pH-Sensitive Polymer-Doxorubicin Conjugate Micelles.

In an effort to prevent metastasis of breast tumor cells- at the same time of inhibiting tumor growth with less toxic side effects, honokiol (HNK) was encapsulated into pH-sensitive polymeric micelles based on the conjugate of poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA) with doxorubicin (DOX), denoted as PEOz-PLA-imi-DOX. PEOz-PLA-imi-DOX was successfully synthesized by connecting DOX to the hydrophobic end of PEOz-PLA via acid-cleavable benzoic imine linker. HNK-loaded conjugate micelles (HNK/PP-DOX-PM) with a size of 21 nm and homogeneous spherical shape exhibited high drug-loading capacity. PEOz-PLA-imi-DOX and HNK/PP-DOX-PM displayed faster release of DOX at pH 5.0 than at pH 7.4. As anticipated, PEOz-PLA-imi-DOX maintained cytotoxicity of DOX against MDA-MB-231 cells. The synergistically enhanced in vitro antitumor effect of HNK/PP-DOX-PM was confirmed by their synergetic inhibition of MDA-MB-231 cell growth. Furthermore, the efficient prevention of tumor metastasis by HNK/PP-DOX-PM was testified by in vitro anti-invasion, wound healing and antimigration assessment in MDA-MB-231 cells, and in vivo bioluminescence imaging in nude mice. The suppression of growth and metastasis of tumor cells by HNK/PP-DOX-PM was attributed to the synergistic effect of pH-triggered drug release and HNK-aroused inhibition of matrix metalloproteinases and epithelial-mesenchymal transition, respectively. In addition, HNK/PP-DOX-PM exhibited superior biosafety than physically encapsulated dual-drug micelles. Consequently, the fabricated HNK/PP-DOX-PM may have great potential for safe and effective suppression of tumor growth and metastasis.

Improved intestinal absorption of paclitaxel by mixed micelles self-assembled from vitamin E succinate-based amphiphilic polymers and their transcellular transport mechanism and intracellular trafficking routes.

To ensure that antitumor drugs can be effectively transported across intestinal barrier and then quickly released in tumor cells, mixed polymeric micelles (Mix-PMs) were designed and fabricated by combining poly(2-ethyl-2-oxazoline)-vitamin E succinate (PEOz-VES) with TPGS1000 for enhancing intestinal absorption of paclitaxel. PEOz-VES exhibited an extremely low critical micelle concentration and negligible cytotoxicity. The Mix-PMs were characterized to have about 20 nm in diameter, uniform spherical morphology, high drug-loading content and sustained drug release profile with a retained pH-sensitivity. The results of the transport through Caco-2 cell monolayers and intestinal absorption revealed that Mix-PMs displayed higher transcellular transport efficiency compared with PEOz-VES micelles and Taxol®. The possible mechanism of transcellular transport for Mix-PMs was elucidated to be mainly through clathrin- and caveolae/lipid rafts-mediated transcytosis. Confocal laser scanning micrographs revealed that late endosomes, lysosomes, endoplasmic reticulum, Golgi apparatus, and mitochondria were all involved in intracellular trafficking of Mix-PMs. The proteins involved in transcytosis of Mix-PMs and finally excreted were unraveled for the first time by the analysis of proteins in the basolateral media according to the proteomics method. Consequently, the fabricated mixed polymeric micelles may have great potential in enhancing intestinal absorption and accelerating drug release in tumor cells.

Concurrently suppressing multidrug resistance and metastasis of breast cancer by co-delivery of paclitaxel and honokiol with pH-sensitive polymeric micelles.

To concurrently suppress multidrug resistance (MDR) and metastasis of breast cancer cells, paclitaxel (PTX) and honokiol (HNK) were coencapsulated into pH-sensitive polymeric micelles based on poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA). The physicochemical properties of dual drug-loaded PEOz-PLA micelles were characterized in size, drug loading and in vitro release. The efficiency of MDR reversal for the micelles was testified by synergetic enhancement of cytotoxicity and uptake by MCF-7/ADR cells. The flow cytometry and fluorescence polarization measurement results reinforced the conclusion that down-regulation of P-gp expression and increase of plasma membrane fluidity appeared to be possible mechanisms of MDR reversal by dual drug-loaded PEOz-PLA micelles. Further, the efficient inhibition of tumor metastasis by dual drug-loaded PEOz-PLA micelles was demonstrated by in vitro anti-invasion and anti-migration assessment in MDA-MB-231 cells and in vivo bioluminescence imaging in nude mice. The suppression of MDR and metastasis by the micelles was assigned to synergistic effects of pH-triggered drug release and HNK/PEOz-PLA-aroused P-gp inhibition, and pH-triggered drug release and PTX/HNK-aroused MMPs inhibition, respectively. In conclusion, our findings strengthen the usefulness of co-delivery of PTX and HNK by pH-responsive polymeric micelles for suppression of tumor MDR and metastasis. STATEMENT OF SIGNIFICANCE: Multidrug resistance (MDR) and metastasis are considered to be two of the major barriers for successful chemotherapy. The combination of a chemotherapeutic drug with a modulator has emerged as a promising strategy for efficiently treating MDR cancer and preventing tumor metastasis. Herein, a dual drug (paclitaxel and honokiol)-loaded pH-sensitive polymeric micelle system based on PEOz-PLA was successfully fabricated to ensure that tumor MDR and metastasis could be concurrently suppressed, therefore achieving distinguishing endo/lysosomal pH from physiological pH by accelerating drug release and then enhancing the cytotoxicity of paclitaxel to drug-resistant tumor cells MCF-7/ADR by increasing cellular uptake of paclitaxel, preventing in vitro invasion and migration for MDA-MB-231 cells and in vivo metastasis in nude mice. Further, the mechanism of MDR reversal by dual drug-loaded PEOz-PLA micelles was elucidated to be down-regulation of P-gp expression and increase of plasma membrane fluidity of MCF-7/ADR cells. The present findings strengthen the usefulness of co-delivery of PTX and HNK by pH-responsive polymeric micelles for suppression of tumor MDR and metastasis.

Mechanisms of pH-Sensitivity and Cellular Internalization of PEOz-b-PLA Micelles with Varied Hydrophilic/Hydrophobic Ratios and Intracellular Trafficking Routes and Fate of the Copolymer.

pH-responsive polymeric micelles have shown promise for the targeted and intracellular delivery of antitumor agents. The present study aimed to elucidate the possible mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles in detail, further unravel the effect of hydrophilic/hydrophobic ratio of the micelles on their cellular internalization, and examine the intracellular trafficking routes and fate of PEOz-b-PLA after internalization of the micelles. The results of variations in the size and Zeta potential of PEOz-b-PLA micelles and cross-sectional area of PEOz-b-PLA molecules with pH values suggested that electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chain at pH lower than its pKa was responsible for pH-sensitivity of PEOz-b-PLA micelles. Furthermore, the studies on internalization of PEOz-b-PLA micelles by MCF-7 cells revealed that the uptake of PEOz-b-PLA micelles was strongly influenced by their structural features, and showed that PEOz-b-PLA micelles with hydrophilic/hydrophobic ratio of 1.7-2.0 exhibited optimal cellular uptake. No evident alteration in cellular uptake of PEOz-b-PLA micelles was detected by flow cytometry upon the existence of EIPA and chlorpromazine. However, the intracellular uptake of the micelles in the presence of MßCD and genistein was effectively inhibited. Hence, the internalization of such micelles by MCF-7 cells appeared to proceed mainly through caveolae/lipid raft-mediated endocytosis without being influenced by their hydrophilic/hydrophobic ratio. Confocal micrographs revealed that late endosomes, mitochondria and endoplasmic reticulum were all involved in the intracellular trafficking of PEOz-b-PLA copolymers following their internalization via endocytosis, and then part of them was excreted from tumor cells to extracellular medium. These findings provided valuable information for developing desired PEOz-b-PLA micelles to improve their therapeutic efficacy and reducing the potential safety risks associated with their intracellular accumulation.

[Transplantation of free latissimus dorsi myocutaneous flaps for craniomaxillofacial reconstruction].

OBJECTIVE: This clinical study was to improve the surgical treatment to craniomaxillofacial tissue defects. METHODS: Since 1997, eight cases with severe craniomaxillofacial defects were treated using free latissimus dorsi myocutaneous flaps. In the operation, nerve anastomosis was performed. Of the 8 cases, 7 were treated in one stage, 1 was treated in 3 steps. The craniomaxillofacial defects ranged from 10 cm x 8 cm to 30 cm x 12 cm. The flaps was 12 cm x 10 cm to 32 cm x 16 cm in size. RESULTS: Postoperative follow-up for 6 months to 4 years demonstrated satisfactory results in all the cases. There was neither necrosis nor ulcer after the operation. The sensation recovery of the flap was also satisfactory. CONCLUSION: Free transfer of the latissimus dorsi myocutaneous flap is an ideal treatment to severe craniomaxillofacial defects as it possesses the advantages of reliable blood supply, ability against infections, large size, concealed donor site, and functional restoration of sensation and movement.