EVA1A reverses lenvatinib resistance in hepatocellular carcinoma through regulating PI3K/AKT/p53 signaling axis.

Lenvatinib is a commonly used first-line drug for the treatment of advanced hepatocellular carcinoma (HCC). However, its clinical efficacy is limited due to the drug resistance. EVA1A was a newly identified tumor suppressor, nevertheless, the impact of EVA1A on resistance to lenvatinib treatment in HCC and the potential molecular mechanisms remain unknown. In this study, the expression of EVA1A in HCC lenvatinib-resistant cells is decreased and its low expression was associated with a poor prognosis of HCC. Overexpression of EVA1A reversed lenvatinib resistance in vitro and in vivo, as demonstrated by its ability to promote cell apoptosis and inhibit cell proliferation, invasion, migration, EMT, and tumor growth. Silencing EVA1A in lenvatinib-sensitive parental HCC cells exerted the opposite effect and induced resistance to lenvatinib. Mechanistically, upregulated EVA1A inhibited the PI3K/AKT/MDM2 signaling pathway, resulting in a reduced interaction between MDM2 and p53, thereby stabilizing p53 and enhancing its antitumor activity. In addition, upregulated EVA1A suppressed the PI3K/AKT/mTOR signaling pathway and promoted autophagy, leading to the degradation of mutant p53 and attenuating its oncogenic impact. On the contrary, loss of EVA1A activated the PI3K/AKT/MDM2 signaling pathway and inhibited autophagy, promoting p53 proteasomal degradation and mutant p53 accumulation respectively. These findings establish a crucial role of EVA1A loss in driving lenvatinib resistance involving a mechanism of modulating PI3K/AKT/p53 signaling axis and suggest that upregulating EVA1A is a promising therapeutic strategy for alleviating resistance to lenvatinib, thereby improving the efficacy of HCC treatment.

Dysfunction of chaperone-mediated autophagy in human diseases.

Chaperone-mediated autophagy (CMA), one of the degradation pathways of proteins, is highly selective to substrates that have KFERQ-like motif. In this process, the substrate proteins are first recognized by the chaperone protein, heat shock cognate protein 70 (Hsc70), then delivered to lysosomal membrane surface where the single-span lysosomal receptor, lysosome-associated membrane protein type 2A (LAMP2A) can bind to the substrate proteins to form a 700 kDa protein complex that allows them to translocate into the lysosome lumen to be degraded by the hydrolytic enzymes. This degradation pathway mediated by CMA plays an important role in regulating glucose and lipid metabolism, transcription, DNA reparation, cell cycle, cellular response to stress and consequently, regulating many aging-associated human diseases, such as neurodegeneration, cancer and metabolic disorders. In this review, we provide an overview of current research on the functional roles of CMA primarily from a perspective of understanding and treating human diseases and also discuss its potential applications for diseases.

TMEM166 inhibits cell proliferation, migration and invasion in hepatocellular carcinoma via upregulating TP53.

Transmembrane protein 166 (TMEM166), an endoplasmic reticulum-associated protein, functions in many diseases via regulating autophagy and/or apoptosis. However, the role of TMEM166 in hepatocellular carcinoma (HCC) remains largely unknown. In this study, we detected the expression of TMEM166 in HCC by real-time fluorescent quantitative PCR (RT-qPCR), immunohistochemistry and western blot. To investigate its biological function and underlying mechanism in HCC, TMEM166 was overexpressed in HCC cell lines and assessed its effects on cell proliferation, migration, invasion, apoptosis and cell cycle by MTT assay, wound healing assay, Transwell assay, Annexin V-FITC/PI assay, JC-1 staining and flow cytometry assay, respectively. Results demonstrated that the expression of TMEM166 was significantly decreased in HCC and was associated with advanced TNM clinical stage and poor clinical outcome of HCC patients. TMEM166 overexpression inhibited HCC cells proliferation, migration and invasion. Furthermore, TMEM166 inhibited cell proliferation by inducing apoptosis and cell cycle arrest via upregulating anti-oncogene TP53 and TP53 knockdown significantly alleviated the anti-tumor effects of TMEM166 on HCC cells. This study provides the first comprehensive analysis the role of TMEM166 in HCC. TMEM166 displays a fine anti-tumor activity on HCC cells involving a mechanism of upregulating TP53. This study suggests TMEM166 is a potential target for the treatment of HCC.

TIM-3 blockade combined with bispecific antibody MT110 enhances the anti-tumor effect of γδ T cells.

As ideal cells that can be used for adoptive cell therapy, γδ T cells are a group of homogeneous cells with high proliferative and tumor killing ability. However, γδ T cells are apt to apoptosis and show decreased cytotoxicity under persistent stimulation in vitro and cannot aggregate at tumor sites efficiently in vivo, both of which are two main obstacles to tumor adoptive immunotherapy. In this study, we found that the immune checkpoint T-cell immunoglobulin domain and mucin domain 3 (TIM-3) were up-regulated significantly on γδ T cells during their ex vivo expansion and this up-regulation contributed to the dysfunction of γδ T cells. Although the killing ability of γδ T cells against breast cancer cells which exhibited a high level of epithelial cell adhesion molecule (EpCAM) was enhanced, the level of TIM-3 on γδ T cells was also further up-regulated under the application of the bispecific antibody MT110 (anti-CD3 × anti-EpCAM) which can redirect T cells to target cells. Besides, these γδ T cells with up-regulated TIM-3 exhibited an increased susceptibility to apoptosis. By reinvigorating dysfunctional γδ T cells and promoting them to accumulate at tumor sites, the combined use of TIM-3 inhibitor and MT110 could further enhance the anti-tumor effect of the adoptively transfused γδ T cells. These results may have clinical implications for the design of new translational anti-tumor regimens aimed at combining checkpoint blockade and immune cell redirection.

Dendritic Cells Therapy with Cytokine-Induced Killer Cells and Activated Cytotoxic T Cells Attenuated Th2 Bias Immune Response.

THE AIM OF THIS STUDY: The purpose of this study is to investigate whether the DC cells combined with CIK cells (DC/CIK) and DC activated cytotoxic T cells (DC-ACT) treatment can promote antitumor response and change the immune indicators by targeting the heterogeneous tumor cell populations at a system level. METHODS: In this study, 112 patients with cancer were assigned to the DC/CIK treatment and 116 patients received the DC-ACT therapy. We detected the lymphocyte subsets and other immune indicators pre- and post-treatment to evaluate the changes of patient's immunity and compare the differences in immune status between two adoptive cellular immunotherapies. RESULTS: DC/CIK therapy elevated the percentage of CD3+ HLA-DR+ T cells, NK cells and several serological cytokines such as IL-2, IL-6 after cell infusion (p < .05). DC-ACT therapy could increase the total CD3 + T cells, CD8 + T cells, CD3+ HLA-DR+ cells and IL-12 cytokines after cell infusion (p < .05). The levels of IL-4/IFN-γ, IL-4/IL-12 and IL-6/IL-12 were reduced significantly in the DC-ACT group compared with DC/CIK group. These observations suggested that DC-ACT therapy has more dominance to induce Th1 cytokine response instead of skewing toward the Th2 cytokine profile based on the immunomodulatory properties. CONCLUSIONS: These results indicated that DC, CIK, and DC-ACT cells exert anti-tumor activity through the different pathways. Thus, this work may provide valuable insights into the clinical curative effect evaluation of immunocyte therapy and the design of combined immunotherapeutic strategies for malignant tumors.

Efficient killing of radioresistant breast cancer cells by cytokine-induced killer cells.

Recurrence of breast cancer after radiotherapy may be partly explained by the presence of radioresistant cells. Thus, it would be desirable to develop an effective therapy against radioresistant cells. In this study, we demonstrated the intense antitumor activity of cytokine-induced killer cells against MCF-7 and radioresistant MCF-7 cells, as revealed by cytokine-induced killer-mediated cytotoxicity, tumor cell proliferation, and tumor invasion. Radioresistant MCF-7 cells were more susceptible to cytokine-induced killer cell killing. The stronger cytotoxicity of cytokine-induced killer cells against radioresistant MCF-7 cells was dependent on the expression of major histocompatibility complex class I polypeptide-related sequence A/B on radioresistant MCF-7 cells after exposure of cytokine-induced killer cells to sensitized targets. In addition, we demonstrated that cytokine-induced killer cell treatment sensitized breast cancer cells to chemotherapy via the downregulation of TK1, TYMS, and MDR1. These results indicate that cytokine-induced killer cell treatment in combination with radiotherapy and/or chemotherapy may induce synergistic antitumor activities and represent a novel strategy for breast cancer.

Design, synthesis, and biological evaluation of anti-EV71 agents.

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), which can spread its infections to the central nervous and other systems with severe consequences. In this article, design, chemical synthesis, and biological evaluation of various anti-EV71 agents which incorporate Michael acceptors are described. Further SAR study demonstrated that lactone type of Michael acceptor provided a new lead of anti-EV71 drug candidates with high anti-EV71 activity in cell-based assay and enhanced mouse plasma stability. One of the most potent compounds (2K, cell-based anti-EV71 EC50=0.028µM), showed acceptable stability profile towards mouse plasma, which resulted into promising pharmacokinetics in mouse via IP administration.

Correction: Zhao, J., et al. Pharmacokinetics of Ginkgolide B after Oral Administration of Three Different Ginkgolide B Formulations in Beagle Dogs. Molecules 2015, 20, 20031-20041.

The authors wish to correct the funding projects number of "Natural Science Foundation of Jiangsu Province" in the Acknowledgments section of this paper [1]: The correct funding projects number should be "No. BK20130403", not "No. BK2013403".[...].

A novel dual EGFR/HER2 inhibitor KU004 induces cell cycle arrest and apoptosis in HER2-overexpressing cancer cells.

Human epidermal growth factor receptor 2 (HER2) is a validated therapeutic target in cancer therapy, and HER2 protein-tyrosine kinase inhibitors have attracted considerable attention in the field of searching for novel anticancer drug candidates. In this study, we investigated the anticancer effect of KU004, a novel dual EGFR and HER2 inhibitor in vitro and in vivo. In vitro, KU004 preferentially inhibited the growth of HER2-overexpressing breast and gastric cell lines and HER2 expression level significantly correlated with response to KU004. It blocked activation of EGFR, HER2 and downstream Akt and Erk and induced G0/G1 arrest which was associated with downregulation of p53, p21, cyclin D1 and CDK4 along with increase of p27 and dephosphorylation of pRb. Apoptosis occurred in a caspase-dependent manner mainly via the extrinsic apoptotic pathway after KU004 treatment. The in vitro efficacy of KU004 was comparable to that of lapatinib. Moreover, KU004 suppressed the growth of NCI-N87 tumor and induced apoptosis without causing apparent weight loss or obvious toxicity. Tumor volume was significantly smaller in KU004-treated group than that in lapatinib-treated group at comparable dose levels. Taken together, these findings demonstrate KU004 can be expected to be a promising anti-HER2 candidate.

Michael acceptor in gambogic acid--Its role and application for potent antitumor agents.

Gambogic acid (GA), a natural product with unique structure, was reported to have broad antiproliferation activities against cancer cell lines. As a reactive Michael acceptor, the 10-position of GA is susceptible to nucleophiles, thus limiting its clinical application as an anticancer agent. Moreover, the 6-OH forms an intramolecular hydrogen bond with 8-CO, which can make the 9, 10 double bond more reactive to nucleophiles. In this essay, two strategies (A and B) were applied to solve the above-mentioned problems. Strategy A was to increase the steric hindrance of C-10 to reduce the activity of GA towards nucleophiles. Strategy B was to replace the hydroxyl of C-6 with other substituents based on the assumption that the intra-molecular hydrogen bond could increase the electrophilicity of C-10. Results showed the electrophilicity of C-10 disappeared as well as the antiproliferation activity against cancer cell lines by introducing a methyl group at C-10. Strategy B showed that the electrophilicity of C-10 was reduced dramatically while maintained the activity by replacement of the hydroxyl of C-6 with neutral or basic groups.

Pharmacokinetics of Ginkgolide B after Oral Administration of Three Different Ginkgolide B Formulations in Beagle Dogs.

Ginkgolide B (GB), an important active constituent of Ginkgo biloba extract, has been used in clinical applications for the treatment of dementia, cerebral insufficiency or related cognitive decline. To investigate the main pharmacokinetic characteristics of three different GB formulations in beagle dogs, a simple, specific and sensitive LC-MS/MS method was established and validated. The separation of the analytes was achieved on an Agilent Eclipse Plus C18 column (1.8 µm, 2.1×50 mm) with a mobile phase consisting of water and acetonitrile. The flow rate was set at 0.4 mL/min. Quantitation was performed using multiple reaction monitoring (MRM) in negative ion mode, with the transitions at m/z (Q1/Q3) 423.1/367.1 for GB and m/z 269.3/170.0 for IS. The linear calibration curve of GB was obtained over the concentration range of 2-200 ng/mL. The intra- and inter-day precisions were <15% and the accuracies were within ±12.7%. The validated method was applied to compare the pharmacokinetic characteristics of GB in healthy beagle dogs after oral administration of three formulations (HME08, GB capsule prepared by hot-melt extrusion technology; LL06, GB pellet prepared by liquid layer technology; conventional GB tablet). The Cmax values of GB from different formulations in beagle dog plasma were 309.2, 192.4 and 66.6 µg/L, and the AUC values were 606.7, 419.1 and 236.2 µg/L·h, respectively. The data suggested that the exposure level of GB from HME08 and LL06 in beagle dog plasma was greatly improved compared with conventional tablets. This study should be helpful for the design and development of oral GB preparations.

[Preparation of hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata and study on its in vitro release mechanism].

In this study, hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata were prepared and the in vitro release behavior were also evaluated. The optimal prescription was achieved by studying the main factor of the type and amount of hydroxypropyl methylcellulose (HPMC) using single factor test and evaluating through cumulative release of three lactones. No burst drug release from the obtained matrix tablets was observed. Drug release sustained to 14 h. The release mechanism of three lactones from A. paniculata was accessed by zero-order, first-order, Higuchi and Peppas equation. The release behavior of total lactones from A. paniculata was better agreed with Higuchi model and the drug release from the tablets was controlled by degradation of the matrix. The preparation of hydrophilic matrix sustained release tablets of total lactones from A. paniculata with good performance of drug release was simple.

[Effects of fenofibrate on the growth and migration of ovarian cancer cells in vitro].

OBJECTIVE: To investigate the effects of fenofibrate, a lipid-lowering drug, on the growth and migration of human ovarian cancer cells SKOV3 in vitro. METHODS: A human ovarian cancer cell line (SKOV3) as the research object, was incubated with serum-free media for 24 h. These cells were then treated by appropriate concentrations of fenofibrate for different time, including control and experimental groups. Cell proliferation was evaluated by MTT assay. Apoptosis was detected by Hoechst/PI and Annexin-V/PI fluorescent assay. The migration of cells was measured by the scratch-wound healing assay. RESULTS: The MTT assay results demonstrated that the fenofibrate (10, 25, 50, 75, 100 micromol/L) could inhibit the proliferation of SKOV3 cells after 24, 48 and 72 h treatment (P < 0.05). The inhibition rate for 24, 48, 72 h-treatment was 55.72% +/- 0.28%, 57.63% +/- 0.47%, 72.41% +/- 0.62% respectively (P < 0.05). The effects increased with the concentrations. Hoechst/PI and Annexin-V/PI fluorescent assay showed that after stimulus for 24 h, fenofibrate induced apoptosis of SKOV3 cells in a concentration-dependent manner was observed. A significant inhibited cells migration distance (P < 0.05) evaluated with scratch-wound healing assay was observed after treatment with fenofibrate (10, 25, 50, 75, 100 micromol/L) for 24 h. CONCLUSION: Lipid-lowering drug fenofibrate can inhibit the growth and migration of human ovarian cancer cell SKOV3 in vitro, to some extent induce apoptosis. But the detailed mechanism need to be further studied.

Piperine suppresses tumor growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model.

AIM: To investigate the effects of piperine, a major pungent alkaloid present in Piper nigrum and Piper longum, on the tumor growth and metastasis of mouse 4T1 mammary carcinoma in vitro and in vivo, and elucidate the underlying mechanisms. METHODS: Growth of 4T1 cells was assessed using MTT assay. Apoptosis and cell cycle of 4T1 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. Real-time quantitative PCR was applied to detect the expression of matrix metalloproteinases (MMPs). A highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model was used to evaluate the in vivo antitumor activity. Piperine was injected into tumors every 3 d for 3 times. RESULTS: Piperine (35-280 µmol/L) inhibited the growth of 4T1 cells in time- and dose-dependent manners (the IC(50) values were 105 ± 1.08 and 78.52 ± 1.06 µmol/L, respectively, at 48 and 72 h). Treatment of 4T1 cells with piperine (70-280 µmol/L) dose-dependently induced apoptosis of 4T1 cells, accompanying activation of caspase 3. The cells treated with piperine (140 and 280 µmol/L) significantly increased the percentage of cells in G(2)/M phase with a reduction in the expression of cyclin B1. Piperine (140 and 280 µmol/L) significantly decreased the expression of MMP-9 and MMP-13, and inhibited 4T1 cell migration in vitro. Injection of piperine (2.5 and 5 mg/kg) dose-dependently suppressed the primary 4T1 tumor growth and injection of piperine (5 mg/kg) significantly inhibited the lung metastasis. CONCLUSION: These results demonstrated that piperine is an effective antitumor compound in vitro and in vivo, and has the potential to be developed as a new anticancer drug.

[Pharmacokinetics of ginkgolides sustained-release tablet].

OBJECTIVE: To study the pharmacokinetics and bioavailability of ginkgolides sustained-release tablet and conventional tablet in Beagle dogs. METHOD: The concentrations of ginkgolides in plasma were determined by LC-MS. The main pharmacokinetic parameters of ginkgolides sustained-release tablet and conventional tablet in vivo were obtained using Pharmacokinetic software DAS 2.0. RESULT: The C(max) of grinkgolide A in ginkgolide sustained-release tablet and conventional tablet were 443.51, 1 039.30 microg x L(-1), respecitvely. t(max) were 2.92, 1.08 h, respectively. AUC(0-12h) were 1 808.21, 2 041.37 h x microg(-1) x L(-1), respectively. MRT were 5.18, 3.18 h, respectively. The relative bioavailability of ginkgolides A was 88.58%. The C(max) of ginkgolide B in ginkgolide sustained-release tablet and conventional tablet were 407.13, 547.38 microg x L(-1), respectively. t(max) were 2.92, 1.08 h, respectively. AUC(01-12 h) were 1 987.31, 1 748.04 h x microg(-1) x L(-1), respectively. MRT were 6.05, 4.98 h, respectively. The relative bioavailability of ginkgolides B was 113.69%. CONCLUSION: The ginkgolides sustained-release tablets have good sustained release characteristics and are bioequivalent to the reference formulation.

EpCAM expression in esophageal cancer and its correlation with immunotherapy of solitomab.

BACKGROUND: Recurrence of esophageal cancer (EC) after chemotherapy may mainly be explained by the existence of chemotherapy-resistant cells, and an effective drug against chemotherapy-resistant cells is highly sought. The aim of this study was to investigate the cytotoxicity of bispecific antibody solitomab combined with γ δ T cells on Eca109 cell spheres. METHODS: We cultured Eca109 cell spheres in serum-free medium, and the morphological differences between wild-type Eca109 cells and Eca109 cell spheres were compared by microscope and flow cytometry. Different concentrations of nanoparticle albumin-bound paclitaxel (Nab-PTX) and cisplatin were used to treat the two groups of cells and compare their drug resistance. Flow cytometry was then used to detect the expression level of epithelial cell adhesion molecule (EpCAM) and the cytotoxicity of γ δ T cells combined with bispecific antibody solitomab on the two groups. RESULTS: Flow cytometry analysis showed that Eca109 cell spheres were smaller in size and had less cytoplasmic granules and CCK-8 assay showed that the viability of Eca109 cell spheres treated with different concentrations of Nab-PTX and cisplatin was significantly higher than that of wild-type Eca109 cells (P<0.05). Flow cytometry also showed that the expression level of EpCAM on Eca109 cell spheres was higher than that of wild-type Eca109 cells. Co-culture experiment showed that there was no significant difference in the cytotoxicity of γ δ T cells to wild-type Eca109 cells and Eca109 cell spheres without solitomab. However, after adding solitomab, the cytotoxicity of γ δ T cells to Eca109 cell spheres was significantly higher than that of wild-type Eca109 cells (P<0.05). CONCLUSIONS: EC Eca109 cell spheres have strong stem cell characteristics such as multidrug resistance and may contain a high proportion of EC stem cells. Further, EC Eca109 cell spheres have a high expression level of EpCAM, and EpCAM may be one of the markers of EC stem cells. Therefore, EpCAM could be used as a potential molecular target of immunotherapy for EC, and solitomab may become an effective immunotherapeutic drug for chemotherapy-resistant EC cells.

Bispecific antibody activated T cells: A newly developed T cells with enhanced proliferation ability and cytotoxicity.

Adoptive cell therapy using ex vivo expanded lymphocytes has shown remarkable efficacy in tumor immunotherapy recently. Among various transfused immune cells, T lymphocytes are the most widely used since they are critical mediators of the immune system and have the capacity to kill tumor cells. However, there are drawbacks in the expanded T cells for transfusion including limited cytotoxicity, limited proliferation and lack of specificity. To improve the quality of these ex vivo expanded T cells, we have designed a new method to expand a group of T cells which are named bispecific antibodies activated T cells. It is the first time that such cells are induced by introducing the bispecific antibody drug (blinatumomab) and feeder cells (normal B cells and irradiated B cell originated lymphoma cells) to the traditional T cells culture system. Culture of freshly isolated human peripheral blood mononuclear cells in this newly designed cell culture system enabled these expanded T cells that (a) displayed a robust proliferation ability; (b) showed fully activated phenotype and enhanced cytokines production; (c) had a low proportion of CD4+CD25+ T regulatory cells and (d) exhibited strengthened cytotoxicity at relatively low effector: target ratios. This work further confirmed the feasibility of rapid induction and expansion of large amounts of human T cells in vitro by using bispecific antibodies and feeder cells. This strategy could also be used for other immune cells rapid expansion and help to improve the quality of these expanded immune cells for adoptive transfusion.

Gemcitabine treatment enhanced the anti-tumor effect of cytokine induced killer cells by depletion of CD4+CD25bri regulatory T cells.

Cytokine induced killer (CIK) cells have a powerful tumor cells killing activity both in vitro and in vivo and transfusion of these cells have become an adjuvant treatment for tumors. CIK cells are induced and amplified from peripheral blood mononuclear cells (PBMCs) with multiple cytokines. As CD4+CD25bri regulatory T cells can be also induced by high dose of interleukin 2 (IL-2) which is used for CIK cells amplification in the CIK cell culture system, the anti-tumor activity of CIK cells was suppressed to some extent. In order to overcome this unwanted suppressive factor, we found that low dose of gemcitabine could reduce the proportion of CD4+CD25bri regulatory T cells in the CIK cell culture system and significantly enhance the anti-tumor activity of CIK cells in vitro. The levels of interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) were also reduced significantly following the depletion of CD4+CD25bri regulatory T cells in gemcitabine treated CIK cell culture system. In vivo experiment showed that low dose of gemcitabine treated CIK cells significantly suppressed tumor growth and prolonged their lifespan in tumor-bearing nude mice, with the proportion of CD4+CD25bri regulatory T cells reduced. Meanwhile, we detected lower levels of IL-10, TGF-ß and a higher level of interferon-γ (IFN-γ) in tumor-bearing nude mice that received gemcitabine treated CIK cells transfusion than those in other groups. The possible mechanism involved in the enhanced anti-tumor activity in vivo was that gemcitabine treated CIK cells created a strengthened anti-tumor immune microenvironment with the changed levels of cytokines such as IL-10, TGF-ß and IFN-γ. These results suggested a strategy to improve the adoptive immune therapy in recent use by removing the suppressive factors and a more effective tumor treatment combining chemotherapy and immunotherapy.

Discovery of Potent EV71 Capsid Inhibitors for Treatment of HFMD.

Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which can spread its infections to the central nervous and other systems with severe consequences. The viral caspid protein VP1 is a well-known target for antiviral efficacy because its occupancy by suitable compounds could stabilize the virus capsid, thus preventing uncoating of virus for RNA release. In this Letter, design, synthesis, and biological evaluation of novel anti-EV71 agents (aminopyridyl 1,2,5-thiadiazolidine 1,1-dioxides) are described. One of the most promising compounds (14) showed excellent antiviral activity against EV71 (EC50 = 4 nM) and exhibited excellent in vivo efficacy in the EV71 infected mouse model.

[Study on Molecular Biology of Rare Two A307 Phenotypes].

OBJECTIVE: To identify the genotypes of the 2 blood samples whose serological typing were difficult by DNA sequencing analysis, and to investigate the molecular genetic basis of their genotypes. METHODS: The 2 blood samples were preliminary genotyped by PCR-SSP. The complete exon 6 and 7 in the ABO genes were amplified by PCR and the PCR products were directly sequenced and clonal sequenced in order to identify the genotypes. RESULTS: The forward typing showed that both samples were weak A, while the reverse typing showed that the samples contained anti-A1. They were preliminarily genotyped as A/O1. RESULTS: The sequencing analysis showed that the 2 samples contained the nt467C>T and nt745C>T mutation in the A allele, which resulted in an amino acid change from Proline (Pro) to Leucine (Leu) at codon 156 and also from Arginine (Arg) to Tryptophan (Trp) at codon 249. CONCLUSION: Through serology results and sequencing analysis, the 2 samples are identified as rare A307 phenotypes.