Rapid screening and identification of anticoagulation component from carthami flos by two-dimensional thrombin affinity chromatography combined with HPLC-MS/MS.

Carthami flos, commonly known as Honghua in China, is the dried floret of safflower and widely acknowledged as a blood stasis promoting herb. The study aimed at investigating the relationship between thrombin and carthami flos through a high-performance thrombin affinity chromatography combined with a high-performance liquid chromatography-tandem mass spectrometry system. First, thrombin was immobilized on the glutaraldehyde-modified amino silica gel to prepare the thrombin affinity stationary phase, which was packed into a small column (1.0 × 2.0 mm, id) for recognizing the anticoagulant active components of carthami flos. The target component was enriched and analyzed by the high-performance liquid chromatography-tandem mass spectrometry system. Finally, hydroxysafflor yellow A was screened out and identified as the active component. The anticoagulant effects of hydroxysafflor yellow A were analyzed by anticoagulant experiments in vitro, and the interaction of hydroxysafflor yellow A with thrombin was investigated by the molecular docking method. The results proved that hydroxysafflor yellow A (30 µg/mL, 0.05 mM) and carthami flos extract (30 µg/mL) could prolong activated partial thrombin time and thrombin time by 50 and 11%, respectively. Moreover, hydroxysafflor yellow A exhibits a good hydrogen bond field and stereo field matching with thrombin. Overall, it was concluded that hydroxysafflor yellow A might exert an anticoagulation effect by interacting with thrombin and thus could be potential anticoagulant drugs for the prevention and treatment of venous thrombosis.

Rational Design of Oxygen Deficiency-Controlled Tungsten Oxide Electrochromic Films with an Exceptional Memory Effect.

Owing to their nonemissive characteristics, electrochromic materials promise distinct advantages in developing next-generation eye-friendly information displays. Yet, it remains a challenge to manipulate the structure of the materials to achieve a strong memory effect with high optical contrast, which is of importance for displaying images with essentially zero energy consumption. Here, we design a mixed crystalline WOx thin film implanted with massive oxygen deficiencies based on a conventional reactive magnetron sputtering process. The obtained WOx film exhibits high dual-band optical modulation in both visible (VIS, 99.0% in 633 nm) and near-infrared (NIR, 94.2% in 1300 nm) regions as well as an exceptional memory effect (the colored transmittance increases only by 0.04% at 633 nm after 50 days). The enhanced electrochromic performance can be attributed to dense Li+-ion binding sites as well as the trapping effect provided by the massive internal oxygen deficiencies. The strategy in this work bestows the WOx thin film a promising candidate for developing electrochromic information displays and other energy-efficient devices as well.

Establishment of thrombin affinity column (TAC)-HPLC-MS/MS method for screening direct thrombin inhibitors from Radix Salviae Miltiorrhiae.

In order to develop an affinity HPLC method for screening direct thrombin inhibitors from Traditional Chinese Medicine (TCM), thrombin was immobilized on the glutaraldehyde-modified amino silica gel and was used as thrombin stationary phase. A thrombin affinity column (TAC) was made by packing the thrombin stationary phase into a bare column (2.0 * 1.0 mm, i.d.). The direct thrombin inhibitors could be screened through this TAC column. For the purpose of improvement of the discovery efficiency, a TAC-HPLC-MS/MS system was used to screen thrombin inhibitors from Radix Salviae Miltiorrhiae (RSM), a famous traditional Chinese medicine. After optimization of all the conditions, cryptotanshinone (Cry), dihydrotanshinone I (Dih-I) and tanshinone IIA (Tan-IIA) were screened out and identified as potential active components. The anticoagulant effects of these three compounds were tested by anticoagulant experiments in vitro. Furthermore, the interaction of three compounds with thrombin was studied by molecular docking. The result shows they have the potential to be used as preventive drugs. In short, this method can be used to screen anticoagulant drugs from traditional Chinese medicine, which provides convenience for screening anticoagulant drugs.

Study on screening potential allergenic proteins from infant milk powders based on human mast cell membrane chromatography and histamine release assays.

Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic (CMC) method based on human mast cells (HMC-1) for screening potential allergens in infant formula milk powders (IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns (10 mm × 2 mm i.d., 5 µm). Under the conditions of 0.2 mL/min flow rate and 214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, ß-casein, α-lactalbumin, and ß-lactoglobulin A, were identified. Furthermore, these proteins and ß-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and ß-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and ß-hexosaminidase. Also, we established a reversed phase liquid chromatographic (RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and ß-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more ß-casein allergic risk than other proteins. This conclusion was consistent with other studies.

The binding of an anti-PD-1 antibody to FcγRΙ has a profound impact on its biological functions.

Antibodies targeting PD-1 have been demonstrated durable anti-cancer activity in certain cancer types. However, the anti-PD-1 antibodies are less or not efficacious in many situations, which might be attributed to co-expression of multiple inhibitory receptors or presence of immunosuppressive cells in the tumor microenvironment. Most of the anti-PD-1 antibodies used in clinical studies are of IgG4 isotype with the S228P mutation (IgG4S228P). The functional impact by the interaction of anti-PD-1 IgG4S228P antibody with Fc gamma receptors (FcγRs) is poorly understood. To assess the effects, we generated a pair of anti-PD-1 antibodies: BGB-A317/IgG4S228P and BGB-A317/IgG4-variant (abbreviated as BGB-A317), with the same variable regions but two different IgG4 Fc-hinge sequences. There was no significant difference between these two antibodies in binding to PD-1. However, BGB-A317/IgG4S228P binds to human FcγRI with high affinity and mediates crosslinking between PD-1 and FcγRI. In contrast, BGB-A317 does neither. Further cell-based assays showed that such crosslinking could reverse the function of an anti-PD-1 antibody from blocking to activating. More importantly, the crosslinking induces FcγRI+ macrophages to phagocytose PD-1+ T cells. In a mouse model transplanted with allogeneic human cancer cells and PBMCs, BGB-A317 showed significant tumor growth inhibition, whereas BGB-A317/IgG4S228P had no such inhibition. Immunohistochemistry study revealed an inverse correlation between FcγRI+ murine macrophage infiltration and the density of CD8+PD-1+ human T cells within tumors in the BGB-A317/IgG4S228P-treated group. These evidences suggested that FcγRI+ binding and crosslinking had negative impact on the anti-PD-1 antibody-mediated anti-cancer activity.

In situ growth of PEDOT/graphene oxide nanostructures with enhanced electrochromic performance.

Poly(3,4-ethylenedioxythiophene) (PEDOT)/graphene oxide (GO) hybrid nanostructures have been obtained by an in situ electro-polymerization process. Field emission scanning electron microscope observation indicates that the hybrid nanostructures consist of uniform and well-dispersed PEDOT nanoparticles integrated on the networked GO nanosheets. Surface chemistry and structure of the hybrid nanostructures have been characterized by X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Electrochemical and optical property measurements demonstrate that the hybrid nanostructures exhibit significantly improved electrochromic performance compared with the pristine PEDOT nanostructure. The contrast between coloring and bleaching state of the PEDOT nanostructure at 480 nm increases from 23.4% to 31.4% after hybridizing with GO nanosheets. The coloring time and bleaching time are shortened from 1800 ms to 300 ms and 1500 ms to 400 ms, respectively, while the coloring efficiency increases from 53.5 cm2 C-1 to 64.9 cm2 C-1 after the hybridization. The obtained PEDOT/GO hybrid nanostructures promise great potential in developing novel electrochromic materials for smart windows and other energy saving applications.

Variation of DNA Methylome of Zebrafish Cells under Cold Pressure.

DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure.

[11'-Deoxyverticillin A induces caspase-dependent cell apoptosis in PC3M cells].

Recent years, the incidence and mortality of prostate cancer have increased dramatically in China. At earlier stages, most diagnosed prostate cancers are responsive to androgen depletion treatment, yet, nearly all patients will eventually progress to metastatic androgen-independent prostate cancer (AIPC), which still has no effective therapeutic method or drug to deal with. 11'-Deoxyverticillin A (C42) belongs to the family of epipolythiodioxopiperazines (ETPs), an interesting class of fungal toxins that inhibit farnesyl transferase. Compounds holding such a property have been explored as putative anticancer agents. In this study, using PC3M cells, an AIPC cell line, we investigated the effect of the compound on apoptosis and explored the underlying mechanism. It revealed that C42 markedly enhanced the activity of caspase-3/7 and increased the accumulation of the cleaved PARP, all of which are the markers of apoptosis. It also revealed that C42 either decreased cell viability or inhibited the growth of PC3M cells. Moreover, we observed that the loss of cell viability and cell growth inhibition induced by C42 were both time- and dosage dependent. Taken together, we indicated that C42 can induce caspase-dependent apoptosis in AIPC cells, and the results presented here will broaden our knowledge about the molecular mechanisms by which C42 exerts its anticancer activity, and future work in this direction may provide valuable information in the development of these compounds into effective cancer therapeutic strategies against androgen-independent prostate cancer.

[Effect of autophagy on expression of interferon in hepatitis B virus-infected hepatocytes].

OBJECT: Autophagy is a lysosomal degradation pathway in which eukaryotic cells dispose intracellular aggregates or defective organelles to maintain cellular homeostasis. Autophagy not only plays a key role in the growth, development, mature and differentiation of cells, but also is associated with pathogenesis, virus infection and immunity. To clarify the mechanism of Hepatitis B virus (HBV) infection and cell immune response, we investigated the relationship between autophagy and IFN factors in the HBV infected cells. METHODS: We inhibited the autophagy by the RNA interference knockdown of Beclin1 and Atg7, the essential autophagic genes, examined the number of autophagosomes by fluorescence microscopy and examined the expression of interferon factors by Real-Time PCR. RESULTS: Autophagy was inhibited after transfected siBeclin1 or siAtg7. After inhibiting the autophagy, the expression of interferon factors were decreased, but cell apoptosis was not induced. CONCLUSION: When the autophagy was inhibited, interferon signaling pathways were impaired in the HBV infected cells. The finding indicated that HBV induced-autophagy enhanced the interferon signaling pathways, and then increased the native immune response.

[Infection with hepatitis B virus enhances basal autophagy].

OBJECTIVE: Hepatitis B virus (HBV) is a major human pathogen that chronically infects 400 million people worldwide. Chronic infection of HBV plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). To clarify the mechanism of HBV-related HCC and immune escape of HBV, we investigated the relationship between infection of HBV and basal autophagy. METHODS: To examine the number of autophagosomes upon transfection with HBV, HepG2.2.15 cells were transfected with green fluorescent protein-microtubule-associated protein 3 (GFP-LC3) and observed by fluorescence microscopy. Huh7 or HepG2 cells was transiently transfected with HBV expression vector pHBV1.3, then the phosphatidylethanol-amine conjugation of microtubule-associated protein 3 (LC3) and the degradation of p62, both of which are specific indictors of autophagy,were evaluated by western blot. Moreover, HepG2 or Chang liver cells were transiently transfected with the constructed HBV X protein ( HBx) expression vector in order to evaluate autophagic status of the cells. RESULTS: HBV and HBx were both able to increase the autophagosomes formation as well as the enhancement of autophagic flux. Notably, C type HBx had a more increment of autophagy than B type does. CONCLUSION: HBV can enhance basal autophagy and the increment is dependent on HBx. Different genotypes of HBx had different effects on basal autophagy. Take together, these findings will help us to clarify the mechanism of HBV infection and the development of Hepatitis B to HCC in the future study.