Efficient Expression of Human Lysozyme Through the Increased Gene Dosage and Co-expression of Transcription Factor Hac1p in Pichia pastoris.

In this work, the high-level expression of the human lysozyme (HLY) was achieved by both optimization of the gene copy number and co-expression of the transcription factor Hac1p for the unfolded protein response (UPR) in the host strain Pichia pastoris KM71H. A series of recombinant constructs with various numbers of HLY expression cassettes was generated for the production of recombinant strains integrated with different copies of the HLY gene. The copy number of the HLY gene was determined by real-time quantitative polymerase chain reaction, and the recombinant strains of P. pastoris carrying one, two, three, four, or six copies of the HLY gene were obtained. Maximum extracellular protein and lysozyme enzyme activity reached 436.99 ± 26.08 µg/mL and 61,900 ± 2036.47 U/mL, respectively, in the recombinant strain HLYH4-3 with the four copies of the HLY gene after shaking flask fermentation. Moreover, the co-expression of the transcription factor Hac1p in the recombinant strains further enhanced the HLY yields. Extracellular protein and lysozyme enzyme activity, respectively, reached 517.82 ± 4.19 µg/mL and 78,600 ± 1134.95 U/mL by using the Hac1p co-expression strain HLYH4-3/Hac1p. These values are the highest recorded level of human lysozyme expressed by P. pastoris in shaking flask fermentation so far.

Bacteria-Loaded Gastro-Retention Oral Delivery System for Alcohol Abuse.

Alcohol abuse is harmful to human health, and many strategies have been developed to retard this harm through protecting liver or activating relative enzymes. In this study, a new strategy of decreasing the alcohol absorption directly depending on the dealcoholization by the bacteria in the upper gastrointestinal (GI) tract was reported. To realize this, a bacteria-loaded gastro-retention oral delivery system with pore structure was constructed through emulsification/internal gelation, which could relieve acute alcohol intoxication in mice successfully. It was found that this bacteria-loaded system kept the above 30% suspension ratio in the simulated gastric fluid for 4 min, displayed good protection effect for the bacteria, and decreased the alcohol concentration from 50 to 30% below within 24 h in vitro. The in vivo imaging results demonstrated that it remained in the upper GI tract until 24 h and reduced 41.9% alcohol absorption. The mice with oral administration of the bacteria-loaded system were found with normal gait, smooth coat, and less liver damage. Although the intestinal flora distribution was influenced slightly during the oral administration, it could restore to normal levels only one day after stopping oral administration quickly, suggesting good biosafety. In conclusion, these results revealed that the bacteria-loaded gastro-retention oral delivery system might intake alcohol molecules rapidly and has huge potential in the treatment of alcohol abuse.

Enhanced Bacterial-Infected Wound Healing by Nitric Oxide-Releasing Topological Supramolecular Nanocarriers with Self-Optimized Cooperative Multi-Point Anchoring.

Polymeric systems that provide cationic charges or biocide-release therapeutics are used to treat the bacteria-infected wound. However, most antibacterial polymers based on topologies with restricted molecular dynamics still do not satisfy the clinical requirements due to their limited antibacterial efficacy at safe concentrations in vivo. Here a NO-releasing topological supramolecular nanocarrier with rotatable and slidable molecular entities is reported to provide conformational freedom to promote the interactions between the carrier and the pathogenic microbes, hence greatly improving the antibacterial performance. With improved contacting-killing and efficient delivery of NO biocide from the molecularly dynamic cationic ligand design, the NO-loaded topological nanocarrier achieves excellent antibacterial and anti-biofilm effects via destroying the bacterial membrane and DNA. MRSA-infected rat model is also brought out to demonstrate its wound-healing effect with neglectable toxicity in vivo. Introducing flexible molecular motions into therapeutic polymeric systems is a general design to enhance the healing of a range of diseases.

A Colon-Targeted Oral Probiotics Delivery System Using an Enzyme-Triggered Fuse-Like Microcapsule.

Probiotics are closely related to human health. However, it is hard to find an appropriate disintegration mode for encapsulation to balance the survival, release, and adhesion of probiotics simultaneously during the current colon-targeted oral delivery, which leads to limited colonization. In this study, an enzyme-triggered fuse-like microcapsule is constructed using alginate and protamine via the electrostatic droplet combined with the layer by layer self-assembly. The multilayer microcapsule can protect the probiotics in the stomach and disintegrate layer by layer under the catalysis of trypsin in the intestine. The formulation with two protamine layers showed the best protection for Escherichia coli MG1655 (EM) during the oral delivery; as well the minimal release at the gastric pH value but a burst release after 1 h at the intestinal pH value. In particular, the adhesion strength of EM is improved with the increase of the layer number. In vivo experiments demonstrate that the EM enters into the stationary phase within 12 h in the colon. Moreover, the blood biochemistry and histological analysis demonstrates the safety of the microcapsule formulation. It can be concluded that this microcapsule can help the probiotics survive during the delivery, then release and colonize in the colon.