Simulation Palynologists for Pollinosis Prevention: A Progressive Learning of Pollen Localization and Classification for Whole Slide Images.

Existing API approaches usually independently leverage detection or classification models to distinguish allergic pollens from Whole Slide Images (WSIs). However, palynologists tend to identify pollen grains in a progressive learning manner instead of the above one-stage straightforward way. They generally focus on two pivotal problems during pollen identification. (1) Localization: where are the pollen grains located? (2) Classification: which categories do these pollen grains belong to? To perfectly mimic the manual observation process of the palynologists, we propose a progressive method integrating pollen localization and classification to achieve allergic pollen identification from WSIs. Specifically, data preprocessing is first used to cut WSIs into specific patches and filter out blank background patches. Subsequently, we present the multi-scale detection model to locate coarse-grained pollen regions (targeting at "pollen localization problem") and the multi-classifiers combination to determine the fine-grained category of allergic pollens (targeting at "pollen classification problem"). Extensive experimental results have demonstrated the feasibility and effectiveness of our proposed method.

HLA-B*27 Heavy Chain Homo-Oligomers Promote the Cytotoxicity of NK Cells via Activation of PI3K/AKT Signaling.

Background and Objectives: Ankylosing spondylitis (AS) is a chronic inflammatory disease and is highly linked with the expression of the human leukocytic antigen-B*27 (HLA-B*27) genotype. HLA-B*27 heavy chain (B*27-HC) has an innate characteristic to slowly fold, resulting in the accumulation of the misfolded B*27-HC and the formation of homo-oligomeric B*27-HC molecules. The homo-oligomeric B*27-HC can act as a ligand of KIR3DL2. Interaction of the homo-oligomeric B*27-HC molecules with KIR3DL2 will trigger the survival and activation of KIR3DL2-positive NK cells. However, the effects of homo-oligomeric B*27-HC molecules associated with KIR3DL2 on the cytotoxic activity of NK cells and their cytokine expressions remain unknown. Materials and Methods: HLA-B*-2704-HC was overexpressed in the HMy2.C1R (C1R) cell line. Western blotting and quantitative RT-PCR were used to analyze the protein expression and cytokine expression, respectively, when C1R-B*-2704 cells that overexpress B*2704-HC were co-cultured with NK-92MI cells. Flow cytometry was used to analyze the cytotoxicity mediated by NK-92MI cells. Results: Our results revealed that NK-92MI cells up-regulated the expression of perforin and enhanced the cytotoxic activity via augmentation of PI3K/AKT signaling after co-culturing with C1R-B*2704 cells. Suppression of the dimerized B*27-HC formation or treatment with an inhibitor of PI3K, LY294002, or with an anti-B*27-HC monoclonal antibody can reduce the perforin expression of NK-92MI after co-culturing with C1R-B*-2704. Co-culturing with C1R-B*-2704 cells suppressed the TNF-α and IL6 expressions of NK-92MI cells. Conclusion: Stimulation of NK cell-mediated cytotoxicity by homo-oligomeric B*27-HC molecules may contribute to the pathogenesis of AS.

Down-Regulation of LOC645166 in T Cells of Ankylosing Spondylitis Patients Promotes the NF-κB Signaling via Decreasingly Blocking Recruitment of the IKK Complex to K63-Linked Polyubiquitin Chains.

Ankylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the spine. AS is highly associated with the expression of HLA-B27. Up to 95% AS patients are HLA-B27-positive. However, only 1%-2% of the HLA-B27-positive carriers suffer from AS, implying that other factors may also govern the development of AS. Long non-coding RNAs (lncRNAs) can regulate the immune response via their binding proteins. In the present study, we have identified that the levels of lncRNA, LOC645166, in T cells of AS patients were reduced. Overexpression of LOC645166 in Jurkat cells down-regulated the IL-23p19 expression and suppressed the JAK2/STAT3 signaling in response to stimulation by phorbol 12-myristate 13-acetate. Suppression of STAT3 activation by LOC645166 was also observed when Jurkat cells or T cells of AS patient were treated with anti-CD3/CD28 antibodies. In order to explore the role of LOC645166 in the pathogenesis of AS, RNA pull-down assay plus proteomic approach and western blotting were performed and identified that LOC645166 prefers binding the K63-linked polyubiquitin chains. LOC645166 can suppress recruitment of the IKK complex to K63-linked polyubiquitin chains and diminish IKK2 activation, leading to down-regulation of NF-κB activation. Down-regulation of LOC645166 expression in T cells of AS patients up-regulates NF-kB activation via decreasingly impeding recruitment of the IKK complex to K63-linked polyubiquitin chains, allowing AS patients to exhibit more sensitivity to stimulation by the proinflammatory cytokines or by TLR ligands.

Identification of phostensin in association with Eps 15 homology domain-containing protein 1 (EHD1) and EHD4.

Phostensin (PTS) encoded by KIAA1949 is a protein phosphatase 1 (PP1)-binding protein. In order to explore the cellular functions of PTS, we have searched PTS-binding proteins by using co-immunoprecipitation in combination with shotgun proteomics. Here, we report two novel PTS-binding proteins, Eps 15 homology domain-containing protein 1 (EHD1) and EHD4. PTS associated with EHD proteins was also observed in GST pull-down assays. Immunofluorescence microscopy demonstrated that the complex was co-localized at the endocytic vesicles. EHD proteins have been known to play a critical role in regulation of endocytic transport. Overexpression of PTS-ß can attenuate the endocytic trafficking of transferrin.

Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis.

We developed a label-free, real-time, and highly sensitive nucleic acid biosensor based on fiber optic particle plasmon resonance (FOPPR). The biosensor employs a single-strand deoxyoligonucleotides (ssDNA) probe, conjugated to immobilized gold nanoparticles on the core surface of an optical fiber. We explore the steric effects on hybridization affinity and limit of detection (LOD), by using different ssDNA probe designs and surface chemistries, including diluent molecules of different lengths in mixed self-assembled monolayers, ssDNA probes of different oligonucleotide lengths, ssDNA probes in different orientations to accommodate target oligonucleotides with a hybridization region located unevenly in the strand. Based on the optimized ssDNA probe design and surface chemistry, we achieved LOD at sub-nM level, which makes detection of target oligonucleotides as low as 1 fmol possible in the 10-mL sensor chip. Additionally, the FOPPR biosensor shows a good correlation in determining HLA-B27 mRNA, in extracted blood samples from patients with ankylosing spondylitis (AS), with the clinically accepted real-time reverse transcription-polymerase chain reaction (RT-PCR) method. The results from this fundamental study should guide the design of ssDNA probe for anti-sense sensing. Further results through application to HLA-B27 mRNA detection illustrate the feasibility in detecting various nucleic acids of chemical and biological relevance.

Esophageal insufflation computed tomography for the diagnosis and management of esophageal submucosal tumors.

BACKGROUND: The selection of therapy for benign esophageal lesions depends in part on whether the lesion extends to or through the esophageal muscle wall. The advent of endoscopic dissection of deep lesions has made this distinction important in the choice between different forms of advanced endoscopic therapy. The goal of this study was to evaluate esophageal insufflation computed tomography (EICT) for the diagnosis and management of esophageal submucosal tumors (SMTs). METHODS: Between April 2011 and May 2013 at the Second Affiliated Hospital of Harbin Medical University, 27 patients with esophageal SMTs diagnosed by gastroscopy were studied observationally. Entry criteria included tumors larger than 0.5 cm. We compared endoscopic ultrasound (EUS) and EICT to assess lesion depth and the relationship between the submucosal lesion and the esophageal wall using the resected lesion as the gold standard. RESULTS: Twenty-seven esophageal SMTs were evaluated. EUS and EICT accurately identified nine as superficial to the muscularis propria. EICT correctly identified the relation of the tumor extension and the outer esophageal wall in all 18 lesions that originated from the muscularis propria; only nine were correctly assessed by EUS (P < 0.001). CONCLUSIONS: EICT enables improved judgment of the relation of esophageal lesions and the esophageal-mediastinal border. We propose EICT as a new, safe, effective, useful, simple and high-tolerance method for assessing the depth and relationships of esophageal submucosal lesions.

Targeted Delivery of the HLA-B∗27-Binding Peptide into the Endoplasmic Reticulum Suppresses the IL-23/IL-17 Axis of Immune Cells in Spondylarthritis.

Ankylosing spondylitis (AS) is highly associated with the expression of human leukocyte antigen-B27 (HLA-B∗27). HLA-B∗27 heavy chain (B27-HC) has an intrinsic propensity to fold slowly, leading to the accumulation of the misfolded B27-HC in the endoplasmic reticulum (ER) and formation of the HLA-B∗27 HC homodimer, (B27-HC)2, by a disulfide linkage at Cys-67. (B27-HC)2 displayed on the cell surface can act as a ligand of the killer-cell Ig-like receptor (KIR3DL2). (B27-HC)2 binds to KIR3DL2 of NK and Th17 cells and activates both cells, resulting in the activation of the IL-23/IL-17 axis to launch the inflammatory reaction in AS patients. However, activation of the IL-23/IL-17 axis originally derived from the HLA-B∗27 misfolding in the ER needs to be characterized. In this study, we delivered two HLA-B∗27-binding peptides, KRGILTLKY and SRYWAIRTR, into the ER by using a tat-derived peptide (GRKKRRQRRR)-His6-ubiquitin (THU) vehicle. Both peptides are derived from the human actin and nucleoprotein of influenza virus, respectively. Our results demonstrated that targeted delivery of both HLA-B∗27-binding peptides into the ER can promote the HLA-B∗27 folding, decrease the levels of (B27-HC)2, and suppress the activation of the IL-23/IL-17 axis in response to lipopolysaccharide. Our findings can provide a new therapeutic strategy in AS.

Characterization of the recognition specificity of BH2, a monoclonal antibody prepared against the HLA-B27 heavy chain.

BH2, a monoclonal antibody prepared against the denatured human leukocytic antigen-B27 heavy chain (HLA-B27 HC), can immunoprecipitate the misfolded HLA-B27 HC complexed with Bip in the endoplasmic reticulum and recognize the homodimerized HLA-B27 HC that is often observed on the cell membrane of patients suffered from ankylosing spondylitis (AS). However, the recognition specificity of BH2 toward the other molecules of HLA-B type and toward the different types of HLA molecules remained uncharacterized. In this study, we carried out the HLA-typing by using the Luminex Technology to characterize the recognition specificity of BH2 and analyzed the binding domain of HLA-B27 HC by BH2. Our results indicated that BH2 preferably binds to molecules of HLA-B and -C rather than HLA-A and the binding site is located within the α2 domain of HLA-B27 HC.

Sulfasalazine Treatment Suppresses the Formation of HLA-B27 Heavy Chain Homodimer in Patients with Ankylosing Spondylitis.

Human leukocytic antigen-B27 heavy chain (HLA-B27 HC) has the tendency to fold slowly, in turn gradually forming a homodimer, (B27-HC)2 via a disulfide linkage to activate killer cells and T-helper 17 cells and inducing endoplasmic reticulum (ER) stress to trigger the IL-23/IL-17 axis for pro-inflammatory reactions. All these consequences lead to the pathogenesis of ankylosing spondylitis (AS). Sulfasalazine (SSA) is a common medication used for treatment of patients with AS. However, the effects of SSA treatment on (B27-HC)2 formation and on suppression of IL-23/IL-17 axis of AS patients remain to be determined. In the current study, we examine the (B27-HC)2 of peripheral blood mononuclear cells (PBMC), the mean grade of sarcoiliitis and lumbar spine Bath Ankylosing Spondylitis Radiology Index (BASRI) scores of 23 AS patients. The results indicated that AS patients without (B27-HC)2 on PBMC showed the lower levels of mean grade of sarcoiliitis and the lumbar spine BASRI scores. In addition, after treatment with SSA for four months, the levels of (B27-HC)2 on PBMCs were significantly reduced. Cytokines mRNA levels, including TNFα, IL-17A, IL-17F and IFNγ, were also significantly down-regulated in PBMCs. However, SSA treatment did not affect the levels of IL-23 and IL-23R mRNAs.

Association between cytokines and methylation of SOCS-1 in serum of patients with ankylosing spondylitis.

In this study, we aim to determine the relationship between methylation level of an inflammatory-related gene, SOCS-1 in serum samples of patients with ankylosing spondylitis (AS) and their degree of inflammation as well as serum cytokine level. Quantitative real time methylation specific PCR was performed to examine the promoter methylation of SOCS-1 in serum samples of 43 HLA-B27+ AS patients and 6 B27+ healthy controls. Degree of inflammation was accessed by spondylopathy, sacroiliitis as well as acute phase reactant, erythrocyte sedimentation rate and C-reactive protein (CRP). Serum IL-6 and TNF-α level was determined by ELISA assay. SOCS-1 methylation can only be found in serums samples from patients but not normal control. Methylation of SOCS-1 significantly associated with severity of patient's spondylopathy (P < 0.005), sacroiliitis (P < 0.005) and acute phase reactant CRP (P = 0.0278). AS patients also exhibited higher serum IL-6 (P < 0.001) and TNF-α level (P < 0.001). Importantly, patients with high serum IL-6 or TNF-α level demonstrated a significantly higher SOCS-1 methylation (P < 0.001). In conclusion, this proof-of-principle study suggested that methylation of SOCS-1 can be detected in serum of HLA-B27+ AS patients but not in B27+ controls. The pathogenic potential of SOCS-1 methylation in AS deserves further investigation.

[Acclimatization and characteristics of microbial community in sulphate-dependent anaerobic methane oxidation].

The greenhouse effect of methane is 26 times worse than that of carbon dioxide, and wastewater containing high concentrations of sulfate is harmful to water, soil and plants. Therefore, anaerobic oxidation of methane driven by sulfate is one of the effective ways for methane reduction. In this paper, with sulfate as the electron accepter, a microbial consortium capable of oxidating methane under anaerobic condition was cultured. The diversity and characteristics of bacterial and archaeal community were investigated by PCR-DGGE, and phylogenetic analysis of the dominant microorganisms was also carried out. The DGGE fingerprints showed that microbial community structure changed distinctly, and the abundance of methane-oxidizing archea and sulfate-reducing bacteria increased in the acclimatization system added sulfate. After acclimatization, the bacterial diversity increased, while archaea diversity decreased slightly. The representative bands in the DGGE profiles were excised and sequenced. Results indicated that the dominant species in the acclimatization system were Spirochaetes, Desulfuromonadales, Methanosarcinales, Methanosaeta. Methane converted into carbon dioxide while sulfate transformed into hydrogen sulfide and sulfur in the process of anaerobic methane oxidation accompanied by sulphate reduction.

Targeted delivery of an antigenic peptide to the endoplasmic reticulum: application for development of a peptide therapy for ankylosing spondylitis.

The development of suitable methods to deliver peptides specifically to the endoplasmic reticulum (ER) can provide some potential therapeutic applications of such peptides. Ankylosing spondylitis (AS) is strongly associated with the expression of human leukocytic antigen-B27 (HLA-B27). HLA-B27 heavy chain (HC) has a propensity to fold slowly resulting in the accumulation of misfolded HLA-B27 HC in the ER, triggering the unfolded protein response, and forming a homodimer, (B27-HC)2. Natural killer cells and T-helper 17 cells are then activated, contributing to the major pathogenic potentials of AS. The HLA-B27 HC is thus an important target, and delivery of an HLA-B27-binding peptide to the ER capable of promoting HLA-B27 HC folding is a potential mechanism for AS therapy. Here, we demonstrate that a His6-ubiquitin-tagged Tat-derived peptide (THU) can deliver an HLA-B27-binding peptide to the ER promoting HLA-B27 HC folding. The THU-HLA-B27-binding peptide fusion protein crossed the cell membrane to the cytosol through the Tat-derived peptide. The HLA-B27-binding peptide was specifically cleaved from THU by cytosolic ubiquitin C-terminal hydrolases and subsequently transported into the ER by the transporter associated with antigen processing. This approach has potential application in the development of peptide therapy for AS.

Identification and characterization of the actin-binding motif of phostensin.

Phostensin, a protein phosphatase 1 F-actin cytoskeleton-targeting subunit encoded by KIAA1949, consists of 165 amino acids and caps the pointed ends of actin filaments. Sequence alignment analyses suggest that the C-terminal region of phostensin, spanning residues 129 to 155, contains a consensus actin-binding motif. Here, we have verified the existence of an actin-binding motif in the C-terminal domain of phostensin using colocalization, F-actin co-sedimentation and single filament binding assays. Our data indicate that the N-terminal region of phostensin (1-129) cannot bind to actin filaments and cannot retard the pointed end elongation of gelsolin-actin seeds. Furthermore, the C-terminal region of phostensin (125-165) multiply bind to the sides of actin filaments and lacks the ability to block the pointed end elongation, suggesting that the actin-binding motif is located in the C-terminal region of the phostensin. Further analyses indicate that phostensin binding to the pointed end of actin filament requires N-terminal residues 35 to 51. These results suggest that phostensin might fold into a rigid structure, allowing the N-terminus to sterically hinder the binding of C-terminus to the sides of actin filament, thus rendering phostensin binding to the pointed ends of actin filaments.

Immunolocalization of phostensin in lymphatic cells and tissues.

Phostensin binds to the pointed ends of actin filaments and modulates actin dynamics. The genomic location of phostensin is between the HLA-C and HLA-E gene clusters on human chromosome 6, and the mRNA of this protein is predominantly distributed in the spleen, thymus, and peripheral leukocytes. However, the distribution of phostensin in leukocyte cell populations and the subcellular localization have not yet been determined. In this study, an anti-phostensin monoclonal antibody (PT2) that recognizes residues 89-124 of phostensin was prepared and used to examine the subcellular localization and distribution of phostensin in white blood cell populations and in lymphatic tissues. It was found that phostensin is mainly concentrated at the cell periphery and co-localizes with actin filaments. In addition, phostensin was abundant in helper T-lymphocytes, cytotoxic T-lymphocytes, mature monocytes, macrophages, B-lymphocytes, natural killer cells, and granulocytes as well as in the lymphatic tissues, such as the thymus, lymph nodes, and spleen. Phostensin is expressed in the mature lymphocytes of the thymic medulla but not in the immature lymphocytes of the thymic cortex. Taken together, phostensin is a ubiquitous protein in leukocytes, and it may play an important role in modulating the cellular functions of leukocytes.

Phostensin caps to the pointed end of actin filaments and modulates actin dynamics.

Phostensin, a protein phosphatase 1 F-actin cytoskeleton targeting subunit encoded by KIAA1949, consists of 165 amino acids and is located between HLA-C and HLA-E gene clusters on human chromosome 6. In this current study, we characterized the biochemical functions of phostensin. Actin dynamics assays using gelsolin-actin seeds showed that phostensin decreases the elongation and depolymerization rates of actin filament pointed ends. The feature of phostensin that binds to the pointed ends of actin filaments was observed through fluorescent single filament binding assay. Taken together, our results suggested that phostensin is an actin filament pointed end-capping protein that is capable of modulating actin dynamics.

HLA haplotype A33-B58-Cw10 may modulate radiographic development of bamboo spine in Taiwanese patients with primary ankylosing spondylitis.

OBJECTIVE: To evaluate the possible relationship between HLA alleles and bony ankylosis of the spine (bamboo spine) in Taiwanese patients with ankylosing spondylitis (AS). METHODS: A small cohort of HLA-B27 positive AS patients was conducted to analyze the effects of alleles {and haplotypes} on the development of bamboo spine. DNA typing of HLA class I and class II genes were performed by SSP method on primary ankylosing spondylitis patients with bamboo spine (n = 84) and spinal enthesopathy controls (n = 228). Odds ratios with 95% confidence intervals and P value were estimated. Determination of the most probable HLA haplotypes on all patients were constructed by comparison of the alleles carried by each patient with the HLA haplotype database established in Taiwanese population studies using homozygosity approach [1] and by expectation-maximum algorithm [2]. RESULTS: Allele frequencies of HLA A33, B58, Cw10, DR4, DR17 and DQ2 were significantly lower in bamboo patients as compared to non-bamboo controls. In contrast, allele frequencies of A24, B54, Cw15, DR11 and DR14 were significantly higher in bamboo patients. Less remarkably, high frequencies of B39, B51, Cw1 and Cw2 alleles were also noted in bamboo patients. Considering linkage disequilibria of alleles in haplotypes, HLA-A11-B27-Cw12 was the most common haplotype in both bamboo and non-bamboo groups (95.23% vs. 91.22%, respectively, P = 0.238). {Haplotypes A33-B58-Cw10, A33-B58-Cw10-DR13 and A33-B58-Cw10-DR17} were significantly lower in bamboo patients as compared to those in controls (P < 0.001, P = 0.001, P = 0.002, respectively). CONCLUSION: Haplotypes A33-B58-Cw10, A33-B58-Cw10-DR13 and A33-B58-Cw10-DR17 showed a strong association with bamboo spine in Taiwanese AS patients. Detection of such haplotypes might be a great aid in the management of patients with the disease.

Increased prevalence of polyomavirus BK viruria that correlates with thrombocytopenia in patients with systemic lupus erythematosus on intensive immunosuppressive therapy.

The prevalence of polyomavirus BK (BKV) reactivation is high in patients with systemic lupus erythematosus (SLE) on long-term immunosuppressants compared to normal population. However, only a few studies are available for the possible correlation of BKV reactivation and clinical manifestations in SLE patients. In the present study, we tried to correlate BKV viruria, clinical manifestations, laboratory findings, and medications in patients with SLE. The urine BKV viral DNA copies were detected from 95 patients with SLE and 32 healthy volunteers by real-time PCR. We found that the prevalence rate of BKV viruria in SLE patients was significantly higher than normal group (71.6% vs. 18.6%, p < 0.001) as well as the urine BKV DNA viral load (4.74 +/- 3.17 vs. 1.08 +/- 2.33 by log scale, p < 0.001). Interestingly, BKV viruria (+) SLE patients had more thrombocytopenic events than BKV viruria ( - ) group (32.4% vs. 3.7%, p = 0.008 after adjustment by age and sex). The patients with BKV viruria DNA copy number >3,200,000/ml exhibited more thrombocytopenia risk than BKV viruria <==3,200,000 copy number/ml or BKV viruria ( - ). The use of potent immunosuppressants may increase BKV viruria. In a refractory thrombocytoponeic case, the add-on of anti-BKV medication, leflunomide 20 mg/day rapidly decreased BKV viruria and recovered platelet counts. In conclusion, our study demonstrated that patients with SLE had higher prevalence rate of BKV reactivation that is correlated with thrombocytopenic episode. Intensive immunosuppressive therapy in SLE may increase the risk of BKV viruria.

Higher LPS-stimulated TNF-alpha mRNA levels in peripheral blood mononuclear cells from Chinese ankylosing spondylitis patients with -308G/A polymorphism in promoter region of tumor necrosis factor: association with distinct A33/B58/Cw10 haplotypes.

To investigate the effects of TNF-alpha -308, -238 promoter polymorphisms on TNF-alpha transcription in B27 positive Chinese patients with ankylosing spondylitis (AS). The possible relationship between polymorphisms, MHC antigens, and quantitative TNF-alpha mRNA expression were evaluated. Single nucleotide polymorphisms (SNPS) of TNF-alpha -308 and -238 were performed by PCR-amplification refractory mutation system method (PCR-ARMS) in sixty-seven B27-positive AS patients and 60 HLA-B27 positive healthy controls in Chinese. Quantitative measurement of TNF-alpha mRNA in peripheral blood mononuclear cells was performed with real time RT-PCR. The polymorphisms were correlated to quantitative TNF-alpha mRNA, and MHC antigens (determined by SSP method) in AS patients. The prevalence rate of both -308G/A and -238G/A TNF-alpha promoter polymorphisms in patients were not significantly different from those in normal subjects. However, a significant high LPS-stimulated TNF-alpha mRNA expression was found in peripheral blood mononuclear cells from patients with promoter -308G/A polymorphism (TNF2) as compared to those in -308G/G genotype (TNF1). Furthermore, -308G/A polymorphism in patients was found to be tightly associated with distinct haplotypes of A33/B58/Cw10 [12 out of 14 -308G/A patients (85.7%) versus none in 53 -308G/G patients], independent of B27 antigen. HLA-A33-B58-Cw10 haplotypes associated TNF-alpha promoter -308G/A polymorphism might play an important role in disease pathogenesis of AS in Chinese population, partially related to a driving force of a higher TNF-alpha production. It confirms once again the importance and complexity of MHC related molecules in disease pathogenesis of AS.

Application of high-speed counter-current chromatography for the isolation of antiviral eremophilenolides from Ligularia atroviolacea.

Ligularia is mainly distributed in the western regions of China. Most of the species have been traditionally used in folk medicine for the treatment of hepatitis B, asthma, hemoptysis and pulmonary tuberculosis. In our continuation of research on antiviral components from traditional Chinese medicine, Ligularia atroviolacea was tested for inhibitory effects on hepatitis B virus (HBV). A bioassay-guided phytochemical examination of L. atroviolacea disclosed that its ethyl acetate extract, which was made up of two eremophilenolides, showed suppressive activity on the expression of HBV surface antigen (HBsAg) in the HepG2.2.15 cell line. Then a simple and effective preparative high-speed counter-current chromatography method was successfully developed for the isolation and purification of two main active metabolites, 8beta-hydroxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide and 8beta-methoxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide from the ethyl acetate extract of L. atroviolacea by a one-step separation using a two-phase solvent system composed of light petroleum (60-90 degrees C)-ethyl acetate-methanol-water (9:1:8:2, v/v/v). The chemical structures of the two eremophilenolides were identified by ESI-MS, (1)H-NMR and (13)C-NMR analysis. The anti-HBV activity of the two purified compounds was measured; both of them showed suppressive activity on the expression of HBsAg in the HepG2.2.15 cell line. The results support the continued and expanded exploitation and utilization of L. atroviolacea.

[Progress in the research of carbon nanotubes as drug carriers].

Research and development of new drug carriers are crucial to the research of drugs. Due to their unique hollow structure and nano-diameter, carbon nanotubes (CNTs) can be used as drug carriers. Functionalization of CNTs with peptides, proteins, nucleic acids or even drug molecules, the so obtained functionalized CNTs can be used as carriers to deliver bioactive molecules into cells without causing any toxicity. The research progress of CNTs as drug carriers in recent years is summarized, and the CNTs' cytotoxicity and their ability to penetrate cells are discussed, and the methods of functionalizing carbon nanotubes are also mentioned in the paper. Along with the advancement of CNTs in drug carriers system, the relationship between the way to functionalize CNTs and the so obtained modified CNTs' ability to penetrate into cells, including the effect of dimension, should be further studied. Preparation of functionalized CNTs with high solubility and low toxicity as drug carriers will be the main research areas in the near future.