Development of a nanobody-based immunoassay for the sensitive detection of fibrinogen-like protein 1.

Immune checkpoint inhibition is an important strategy in cancer therapy. Blockade of CTLA-4 and PD-1/PD-L1 is well developed in clinical practice. In the last few years, LAG-3 has received much interest as an emerging novel target in immunotherapy. It was recently reported that FGL1 is a major ligand of LAG-3, which is normally secreted by the liver but is upregulated in several human cancers. FGL1 is a crucial biomarker and target for cancer immunotherapy. As the efficacy of immunotherapy is limited to specific types of patients, the subset of patients needs to be selected appropriately to receive precise treatment according to different biomarkers. To date, there is no test to accurately assess FGL1 expression levels. Nanobodies have some outstanding features, such as high stability, solubility and affinity for diagnostic and therapeutic applications. Here, we report the development and validation of a rapid, sensitive, and cost-effective nanobody-based immunoassay for the detection of FGL1 in human serum. In this study, human FGL1 recombinant protein was expressed and purified for the first time as an immunized antigen. Then, we constructed a nanobody phage display library and screened several nanobodies that bind FGL1 with high affinity. We selected two nanobodies targeting different epitopes of FGL1, one as a capture and the other conjugated with HRP as a probe. The double nanobody-based sandwich ELISA to detect the concentration of FGL1 showed a good response relationship in the range of 15.625-2000 ng/mL, and the recoveries from the spiked sample were in the range of 78% and 100%. This assay could be used as a potential approach for evaluating FGL1 expression for patient stratification and for predicting the therapeutic efficacy of targeting the LAG3/FGL1 axis.

[Identification on Xanthium sibiricum from different habitats by FTIR fingerprint patterns].

OBJECTIVE: To establish an FTIR method to identify Xanthium sibiricum from different habitats. METHODS: FTIR spectra of Xanthium sibiricum from different habitats were analyzed,and the similarity of different fingerprint spectra and the chemical pattern recognition were calculated and analyzed according to the wave numbers of peaks. RESULTS: Different FTIR spectra of 10 different habitats of Xanthium sibiricum were obtained,and the similarities were all above 0. 96. CONCLUSION: This method can be used for identification on Xanthium sibiricum from different habitats. The results of similarity calculation and chemical pattern recognition further prove the feasibility of this method.

[Identification on Solanum lyratum by FT-IR fingerprint patterns and similarity degree of different fingerprint patterns].

OBJECTIVE: To provide a FT-IR new method to identify different habitats of Solanum lyratum. METHODS: Analyzed FT-IR patterns of Solanum lyratum from different habitats, and the similarity of different fingerprint patterns was calculated and analyzed according to the wave numbers of peaks searched. RESULTS: Obtained the different FT-IR pattern of 5 different habitats of Solanum lyraturn. CONCLUSION: This method can be used for identifications on different habitats of Solanum lyratum. The results of similarity calculation further prove the feasibility of this method.

[Identification on Solanum lyratum by X-ray diffraction Fourier fingerprint and similarity analysis].

OBJECTIVE: To provide an X-ray diffraction (XRD) method for identifying different medicinal parts of Solanum lyratum. METHODS: Analyzed X-ray diffraction Fourier patterns of different parts of Solanum lyratum, and the similarity degree of different fingerprint was calculated and analyzed according to the position (2 theta value)of peaks searched. RESULTS: Different X-ray diffraction Fourier patterns of different medicinal parts of Solanum lyratum were obtained. CONCLUSION: This method can be used for identifying different medicinal parts of Solanum lyratum. The results of similarity calculation further proves the feasibility of this method.

The tissue distribution in mice and pharmacokinetics in rabbits of oxaliplatin liposome.

A rapid, sensitive, and simple high-performance liquid chromatographic (HPLC) method with an ultraviolet detector (UV) has been developed for the determination of oxaliplatin in the plasma of rabbits and tissues of mice. The sample preparation was carried out by complexation with 0.5 mL of DETC (diethyl-dithiocarbamate) solution and extracted by ether and chloroform. Then, 20 microL of supernatant was injected into the HPLC system with 0.25 mol/L of sodium chloride solution and methanol (30:70 v/v) as the mobile phase at a flow rate of 1.0 mL/min. Separation was performed with a C(18) column at 25 degrees C. The peak was detected at 254 nm. The calibration curve was linear (R(2) > or = 0.9995) in the concentration range of 0.1 approximately 200 microg/mL in plasma and tissues. The intra- and interday variation coefficients were not more than 2.61 and 3.83%, respectively. The limit of detection was 20 ng/mL. The mean recoveries of oxaliplatin were ranged from 97.83 to 104.17% in plasma and tissues. The present method has been successfully applied to the pharmacokinetic study of oxaliplatin liposome in mice and rabbits.

A conductive sodium alginate and carboxymethyl chitosan hydrogel doped with polypyrrole for peripheral nerve regeneration.

Polymer materials with electrically conductive properties have good applications in their respective fields because of their special properties. However, they usually exhibited poor mechanical properties and biocompatibility. In this work, we present a simple approach to prepare conductive sodium alginate (SA) and carboxymethyl chitosan (CMCS) polymer hydrogels (SA/CMCS/PPy) that can provide sufficient help for peripheral nerve regeneration. SA/CMCS hydrogel was cross-linked by calcium ions provided by the sustained release system consisting of d-glucono-δ-lactone (GDL) and superfine calcium carbonate (CaCO3), and the conductivity of the hydrogel was provided by doped with polypyrrole (PPy). Gelation time, swelling ratio, porosity and Young's modulus of the conductive SA/CMCS/PPy hydrogel were adjusted by polypyrrole content, and the conductivity of it was within 2.41 × 10-5 to 8.03 × 10-3 S cm-1. The advantages of conductive hydrogels in cell growth were verified by controlling electrical stimulation of cell experiments, and the hydrogels were also used as a filling material for the nerve conduit in animal experiments. The SA/CMCS/PPy conductive hydrogel showed good biocompatibility and repair features as a bioactive biomaterial, we expect this conductive hydrogel will have a good potential in the neural tissue engineering.

[Study on the inclusion technology of volatile oil in Shiquandabu pills].

OBJECTIVE: To extract volatile oil from Shiquandabu pills and study technological process of its inclusion compound with beta-Cyclodextrin (beta-CD) to improve its stability. METHODS: The study was carried out with steam distillation and orthogonal design. The process conditions were studied by determining the utilization and ratio of oil from Shiquandabu pills volatile oil and extract ratio of in-clusion compound. The most important factor was the ratio of oil to beta-CD. and the inclusion compound was identified by FT-IR spectra and XRD. RESULTS: The optimum preparation conditions for inclusion were best-established as oil: beta-CD was 1:8, the inclusion time and temperature were for 1.0 hours. at 20 degrees C. CONCLUSION: Tne method can be used for increasing volatile oil's stability and its solubility. It is suitable for production of medicinal preparation.

[Identification on Spora Lygodii by X-ray diffraction fingerprint spectra method].

OBJECTIVE: To set up a new identification and analysis method of Spora Lygodii. METHOD: To get the extracts of Spora Lygodii using methanol, chloroform and petroleum ether as solvent and the extracts were identified, analyzed by X-ray diffraction Fourier fingerprint spectra. RESULTS: Experiments and analysis were carried out on three samples. The standard X-ray diffraction Fourier fingerprint spectra and characteristic diffraction peaks were obtained. There were some differences among the spectra of the extracts, but the characteristic diffraction peaks were obvious. CONCLUSION: The experimental result indicated that X-ray diffraction Fourier fingerprint spectra can be used to identify and analyze the Chinese traditional herb Lygodium japonicum (Thunb.) Sw.

Preparation, characterization and in vitro release study of a glutathione-dependent polymeric prodrug Cis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan.

In this work, an amphiphilic polymeric prodrug Cis-3-(9H-purin-6-ylthio)-acrylic acid-graft-carboxymethyl chitosan (PTA-g-CMCS) was designed and synthesized. In aqueous solution, this grafted polymer can self-assemble into spherical micelles with a size ranging from 104 to 285 nm and zeta potential ranging from -12.3 to -20.1 mV. For the release study, less than 24% of 6-Mercaptopurine (6-MP) was released from PTA-g-CMCS1 in the media containing 2 and 100 µM glutathione (GSH), whereas 37%, 54% and 75% of 6-MP was released from the media with GSH of 1, 2 and 10mM, respectively. Besides, pH and drug content of the polymeric prodrug only presented slight influence on the 6-MP release. MTT assay demonstrated that this system had higher inhibition ratio on HL-60 cells (human promyelocytic leukemia cells) in the presence of GSH and lower cytotoxicity on mouse fibroblast cell line (L929). Therefore, this nano-sized system is glutathione-dependent, and it can be employed as a potential carrier for the controlled release of 6-MP.

In vitro and in vivo evaluation of the wound healing properties of Siegesbeckia pubescens.

ETHNOPHARMACOLOGICAL RELEVANCE: Siegesbeckia pubescens (SP) has been traditionally used as a wound healing agent. AIM OF THE STUDY: Investigate in vitro and in vivo healing properties of SP extract. MATERIALS AND METHODS: The methanolic extract of SP was tested for the ability to stimulate the growth of mouse fibroblast NIH3T3 in vitro. The viability and proliferation of fibroblasts were evaluated at 72 h after cell seeding by MTT assay at 570 nm. To study wound healing properties in vivo, excision and incision wound models were used on rats and SP (3, 4, 5%, w/w) was topically administered. After treatment, wound contraction, epithelialization period and hexosamine content were evaluated in the excision wound model. In the incision wound model, wound sites were removed for histopathological analysis and skin-breaking strength determination. RESULTS: The methanol extract showed significant stimulation of the growth of mouse fibroblast NIH3T3 at 0.5-100 µg/mL. In excision wound, animals treated with 4, 5% (w/w) SP exhibited significant increases in the rate of wound contraction, period of epithelialization and content of hydroxyproline. In incision wound, the animals treated with both the 4 and 5% (w/w) SP extracts showed an increase in breaking strength when compared with the control, which was additionally supported by histopathological studies. CONCLUSION: The experimental data revealed that the methanolic extract of SP displayed remarkable wound healing activity, corroborating its traditional use.

Topical anti-inflammatory and analgesic activity of kirenol isolated from Siegesbeckia orientalis.

ETHNOPHARMACOLOGICAL RELEVANCE: Siegesbeckia orientalis has been traditionally used as a topical anti-inflammatory and analgesic agent. AIMS OF THE STUDY: Current study was designed to explore the topical anti-inflammatory and analgesic effects of a constituent isolated from Siegesbeckia orientalis (Compositae), in order to validate its folk use. MATERIALS AND METHODS: Kirenol was isolated from ethanolic extract of Siegesbeckia orientalis. Several topical formulations containing kirenol were investigated for anti-inflammatory and analgesic activities in rat. The effects were studied using carrageenan-induced rat acute inflammation model, complete Freund's adjuvant (CFA)-induced chronic inflammation and formalin test in rats. Piroxicam gel and methyl salicylate ointment were studied as positive control for anti-inflammatory and analgesic activity, respectively. RESULTS: The anti-inflammatory effect of kirenol 0.4-0.5% (w/w) was similar to the effect of piroxicam gel 4h after carrageenan injection. The analgesic activity of topical preparation with more than 0.4% (w/w) was observed in the late phase. These effects may be due, at least in part, to the pro-inflammatory cytokine production of IL-1ß and TNF-α. The administration of kirenol cream at the dose of 0.3, 0.4 and 0.5% (w/w) significantly inhibited the development of joint swelling induced by CFA, which was auxiliary supported by histopathological studies. CONCLUSION: Kirenol has demonstrated its significant potential to be further investigated for its discovery as a new lead compound for management of topical pain and inflammation, although further pharmacological research is necessary to fully understand its mechanism of action. It also supports the potential beneficial effect of topically administered Siegesbeckia orientalis in inflammatory diseases.