RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Anti-colorectal cancer effects of anti-p21Ras scFv delivered by the recombinant adenovirus KGHV500 and cytokine-induced killer cells.

BACKGROUND: Colorectal cancer (CRC) is the most common type of gastrointestinal cancer. CRC gene therapy mediated by adenovirus holds great promise for the treatment of malignancies. However, intravenous delivery of adenovirus exhibits limited anti-tumor activity in vivo when used alone. METHODS: In this study, the antitumor activity of the recombinant adenovirus KGHV500 was assessed with the MTT, TUNEL, Matrigel invasion and cell migration assays. To enhance the intravenous delivery of KGHV500 in vivo, cytokine-induced killer (CIK) cells were used as a second vector to carry KGHV500. We explored whether CIK cells could carry the recombinant adenovirus KGHV500 containing the anti-p21Ras single chain fragment variable antibody (scFv) gene into tumors and enhance antitumor potency. RESULTS: Our results showed that KGHV500 exhibited significant antitumor activity in vitro. In the nude mouse SW480 tumor xenograft model, the combination of CIK cells with KGHV500 could induce higher antitumor activity against colorectal cancer in vivo than that induced by either CIK or KGHV500 alone. After seven days of treatment, adenovirus and scFv were detected in tumor tissue but were not detected in normal tissues by immunohistochemistry. Therefore, KGHV500 replicates in tumors and successfully expresses anti-p21Ras scFv in a colorectal cancer xenograft model. CONCLUSIONS: Our study provides a novel strategy for the treatment of colorectal cancer by combining CIK cells with the recombinant adenovirus KGHV500 which carried anti-p21 Ras scFv.

Research progress on the structure, derivatives, pharmacological activity, and drug carrier capacity of Chinese yam polysaccharides: A review.

Chinese yam is a traditional Chinese medicine that has a long history of medicinal and edible usage in China and is widely utilised in food, medicine, animal husbandry, and other industries. Chinese yam polysaccharides (CYPs) are among the main active components of Chinese yam. In recent decades, CYPs have received considerable attention because of their remarkable biological activities, such as immunomodulatory, antitumour, hypoglycaemic, hypolipidaemic, antioxidative, anti-inflammatory, and bacteriostatic effects. The structure and chemical alterations of polysaccharides are the main factors affecting their biological activities. CYPs are potential drug carriers owing to their excellent biodegradability and biocompatibility. There is a considerable amount of research on CYPs; however, a systematic summary is lacking. This review summarises the structural characteristics, derivative synthesis, biological activities, and their usage as drug carriers, providing a basis for future research, development, and application of CYPs.

Robust Osteoconductive ß-Tricalcium Phosphate/L-poly(lactic acid) Membrane via Orientation-Strengthening Technology.

L-poly(lactic acid) (PLLA) is a biodegradable material with multiple biomedical application potentials, especially as a membrane for guided bone regeneration. In terms of its low strength and poor osteogenic activity, improving these two properties is the key to resolve the limitations of PLLA for bone-associated applications. Herein, an orientation-strengthening technology (OST) was developed to reinforce PLLA's mechanical strength by introducing biocompatible ß-tricalcium phosphate (ß-TCP) to improve the crystallinity of PLLA, allowing for the formation of a highly oriented architecture to acquire an advanced membrane with high mechanical property. Furthermore, the addition of ß-TCP nanoparticles significantly promotes the osteogenic activity of the composites. The tensile strength of the membrane containing 5 wt % ß-TCP was 220 MPa, which was 4-folds that of the native polylactic acid fabricated via the conventional method. The oriented microstructure enhanced both the mechanical strength and the osteogenic activity of the material. The parallel grooves on the material surface are similar to the mineralized collagen fibers on the bone surface, which promoted the growth and differentiation of osteoblasts, with ß-TCP further contributing to the osteoconductive effect. The combination of ß-TCP and orientation-strengthening effect endows the material with higher mechanical properties and bioactivities, which provides an advanced manufacturing strategy for the preparation of PLLA-based materials for bone repair.

Effects of Process Parameters on the Quality of Suantang Beef.

Suantang beef is a traditional delicious Chinese food cooked in Suantang (ST, a sour soup fermented by microorganisms). However, the impact of ST on beef quality is unclear, and the process of ST beef lacks unified technical standards. In the presented study, we found that the additional amount of salt, cooking time, meat thickness, and beef-ST ratio significantly affect the quality of ST beef. After optimization, it was found that when salt addition was 1%, cooking time was 3 min, meat thickness was 2 cm, and beef-ST ratio was 40%, the color determined by colorimeter, texture determined by texture analyzer, and sensory scores of beef cooked by ST were improved compared with boiled beef. ST decreased the pH value and cathepsin L activity of beef, increased the content of organic acid, and changed the protein composition of beef. ST made the beef have higher hardness, and have better chewiness and cohesion. At the same time, ST reduced the disagreeable odors of beef and improved beef flavor. In addition, 88 volatile compounds were detected in ST beef by HS-SPME/GC-MS. According to odor, threshold, and odor activity value (OAV), 24 critical aroma-active compounds were confirmed in ST beef. This study provides a basis for the potential industrialized production of ST beef.

Biomechanical Effects of a Cross Connector in Sacral Fractures - A Finite Element Analysis.

Background: Spinopelvic fractures and approaches of operative stabilization have been a source of controversial discussion. Biomechanical data support the benefit of a spinopelvic stabilization and minimally invasive procedures help to reduce the dissatisfying complication rate. The role of a cross connector within spinopelvic devices remains inconclusive. We aimed to analyze the effect of a cross connector in a finite element model (FE model). Study Design: A FE model of the L1-L5 spine segment with pelvis and a spinopelvic stabilization was reconstructed from patient-specific CT images. The biomechanical relevance of a cross connector in a Denis zone I (AO: 61-B2) sacrum fracture was assessed in the FE model by applying bending and twisting forces with and without a cross connector. Biomechanical outcomes from the numerical model were investigated also considering uncertainties in material properties and levels of osseointegration. Results: The designed FE model showed comparable values in range-of-motion (ROM) and stresses with reference to the literature. The superiority of the spinopelvic stabilization (L5/Os ilium) ± cross connector compared to a non-operative procedure was confirmed in all analyzed loading conditions by reduced ROM and principal stresses in the disk L5/S1, vertebral body L5 and the fracture area. By considering the combination of all loading cases, the presence of a cross connector reduced the maximum stresses in the fracture area of around 10%. This difference has been statistically validated (p < 0.0001). Conclusion: The implementation of a spinopelvic stabilization (L5/Os ilium) in sacrum fractures sustained the fracture and led to enhanced biomechanical properties compared to a non-reductive procedure. While the additional cross connector did not alter the resulting ROM in L4/L5 or L5/sacrum, the reduction of the maximum stresses in the fracture area was significant.