Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell acti-vators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside Ⅳ,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory compo-nents of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

OBJECTIVETo study the repair effect of low intensity pulsed ultrasound (LIPUS) irradiating combined with guided bone regeneration (GBR) on the defect of Beagle dog canines periodontal bone.

METHODSFour canine teeth of every beagle dog (8 beagle dogs) were randomly distributed: Group 1 (LIPUS disposal + GBR+ autogenous bone graft group), group 2 (LIPUS disposal + autogenous bone graft group), group 3 (GBR + autogenous bone graft group), blank control group. The model of periodontal bone defect was established in the 1/3 part of the root buccal area. According to the group division, autogenous bone were grafted, group 1 and group 2 were disposed by LIPUS 20 min x d(-1). The intensity of ultrasound were 30 mW x cm(-2). Group 1 and group 3 were injected with Bio-Gide collagen membrane. The beagle dogs were executed at 6 and 8 weeks of the disposal of LIPUS and then Micro-CT test and analysis were conducted to periodontal bone defect area of each group.

RESULTSBy clinical observation, there were different degrees of shrinkages of the area of periodontal bone defect in each group. The Micro-CT test analysis indicated that there was a statistical difference among the number, the thickness as well as the size of bone trabecula of each group (P < 0.05). However, there was no statistical difference (P > 0.05) in terms of bone issue measurement index between 6 to B weeks of each group. Group 1 had the most new bone.

CONCLUSIONLIPUS has the potential to promote the repair of periodontal bone defect. Therefore the combination of LIPUS and GBR may be more conducive to the repair and regeneration of periodontal bone defect.

Animals ; Bone Regeneration ; Bone Transplantation ; Bone and Bones ; Collagen ; Dogs

Biochromatography is a new chromatographic technology with great development potential. It has been widely used in drug screening and biomolecular interaction analysis. The core of this technology is the chromatographic stationary phase of biomolecules. Nowadays, it mainly develops cell membrane chromatography, artificial biomimetic membrane chromatography and the various immobilization strategies to directly immobilizes proteins on the stationary phase carrier. This paper reviews the research progress of new biochromatographic stationary phase and the application of biochromatographic analysis based on new stationary phase. And, the applications of biochromatographic stationary phase and micro biochromatographic analysis system based on monolithic column are prospected.

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with K _D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl₄-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

RAS, a member of the small GTPase family, functions as a binary switch by shifting between inactive GDP-loaded and active GTP-loaded state. RAS gain-of-function mutations are one of the leading causes in human oncogenesis, accounting for ∼19% of the global cancer burden. As a well-recognized target in malignancy, RAS has been intensively studied in the past decades. Despite the sustained efforts, many failures occurred in the earlier exploration and resulted in an 'undruggable' feature of RAS proteins. Phosphorylation at several residues has been recently determined as regulators for wild-type and mutated RAS proteins. Therefore, the development of RAS inhibitors directly targeting the RAS mutants or towards upstream regulatory kinases supplies a novel direction for tackling the anti-RAS difficulties. A better understanding of RAS phosphorylation can contribute to future therapeutic strategies. In this review, we comprehensively summarized the current advances in RAS phosphorylation and provided mechanistic insights into the signaling transduction of associated pathways. Importantly, the preclinical and clinical success in developing anti-RAS drugs targeting the upstream kinases and potential directions of harnessing allostery to target RAS phosphorylation sites were also discussed.