A Universal Cyclodextrin-Based Nanovaccine Platform Delivers Epitope Peptides for Enhanced Antitumor Immunity.

Currently, there is still an intense demand for a simple and scalable delivery platform for peptide-based cancer vaccines. Herein, a cyclodextrin-based polymer nanovaccine platform (CDNP) is designed for the codelivery of peptides with two immune adjuvants [the Toll-like receptor (TLR)7/8 agonist R848 and the TLR9 agonist CpG] that is broadly applicable to epitope peptides with diverse sequences. Specifically, the cyclodextrin-based polymers are covalently linked to epitope peptides via a bioreactive bond-containing cross-linker (PNC-DTDE-PNC) and then physically load with R848 and CpG to obtain CDNP. The CDNP efficiently accumulats in the lymph nodes (LNs), greatly facilitating antigen capture and cross-presentation by antigen-presenting cells. The immunogenicity of the epitope peptides is significantly enhanced by the codelivery and synergy of the adjuvants, and the CDNP shows the ability to inhibit tumor progression in diverse tumor-bearing mouse models. It is concluded that CDNP holds promise as an optimized peptide-based cancer vaccine platform.

Long noncoding RNA CR46040 is essential for injury-stimulated regeneration of intestinal stem cells in Drosophila.

Long noncoding RNAs (lncRNAs) play important regulatory roles in stem cell self-renewal, pluripotency maintenance, and differentiation. Till now, there is very limited knowledge about how lncRNAs regulate intestinal stem cells (ISCs), and lncRNAs mediating ISC regeneration in Drosophila have yet been characterized. Here, we identify a lncRNA, CR46040, that is essential for the injury-induced ISC regeneration in Drosophila. Loss of CR46040 greatly impairs ISC proliferation in response to tissue damage caused by dextran sulfate sodium (DSS) treatment. We demonstrate that CR46040 is a genuine lncRNA that has two isoforms transcribed from the same transcription start site and works in trans to regulate intestinal stem cells. Mechanistically, CR46040 knock-out flies failed to fully activate JNK, JAK/STAT, and HIPPO signaling pathways after tissue damage, which are required for ISC proliferation after intestinal injury. Moreover, CR46040 knock-out flies are highly susceptible to DSS treatment and enteropathogenic bacteria Erwinia carotovora ssp. carotovora 15 (Ecc15) infection. Our findings characterize, for the first time, a lncRNA that mediates damage-induced ISC proliferation in Drosophila and provide new insights into the functional links among the long noncoding RNAs, ISC proliferation, and tissue homeostasis.

Profiling ATM regulated genes in Drosophila at physiological condition and after ionizing radiation.

BACKGROUND: ATM (ataxia-telangiectasia mutated) protein kinase is highly conserved in metazoan, and plays a critical role at DNA damage response, oxidative stress, metabolic stress, immunity, RNA biogenesis etc. Systemic profiling of ATM regulated genes, including protein-coding genes, miRNAs, and long non-coding RNAs, will greatly improve our understanding of ATM functions and its regulation.  RESULTS: 1) differentially expressed protein-coding genes, miRNAs, and long non-coding RNAs in atm mutated flies were identified at physiological condition and after X-ray irradiation. 2) functions of differentially expressed genes in atm mutated flies, regardless of protein-coding genes or non-coding RNAs, are closely related with metabolic process, immune response, DNA damage response or oxidative stress. 3) these phenomena are persistent after irradiation. 4) there is a cross-talk regulation towards miRNAs by ATM, E2f1, and p53 during development and after irradiation. 5) knock-out flies or knock-down flies of most irradiation-induced miRNAs were sensitive to ionizing radiation. CONCLUSIONS: We provide a valuable resource of protein-coding genes, miRNAs, and long non-coding RNAs, for understanding ATM functions and regulations. Our work provides the new evidence of inter-dependence among ATM-E2F1-p53 for the regulation of miRNAs.

Co-delivery of proanthocyanidin and mitoxantrone induces synergistic immunogenic cell death to potentiate cancer immunotherapy.

Immunological checkpoint inhibitors provide a revolutionary method for cancer treatment. However, due to low tumor mutations and insufficient infiltration of immune cells into the tumor microenvironment, 85% of colorectal cancer patients cannot respond to checkpoint blockade immunotherapy. In this study, tumor microenvironment-responsive deformable nanoparticles (DMP@NPs) were rationally designed to improve immunotherapy by synergistically modulating the immune tumor microenvironment. DMP@NPs self-assemble from a newly synthesized tumor acidity responsive polypeptide checkpoint inhibitor polymer (PEG-DMA-DPPA-1) with immunogenic cell death (ICD) enhanced combination drugs containing a certain proportion of mitoxantrone (MITX) and proanthocyanidins (PC). Upon tumor acidity-triggered cleavage of PEG-DMA-DPPA-1, DMP@NPs undergo special "sphere-ring deformation" dissociation, gradually releasing polypeptide checkpoint inhibitor DPPA-1, MITX and PC. MITX/PC in vitro synergistically triggers higher ICD with the release of the high mobility group box-1 (HMGB-1) and calreticulin (CRT). After intravenous injection of DMP@NPs, the local tumor microenvironment of CT26 tumor-bearing mice was reprogrammed, and dendritic cell activation and T cell infiltration were significantly increased. Most importantly, the synergistic immune nanodrug DMP@NPs improved the efficacy of colorectal cancer immunotherapy and reduced toxicity and side effects for the immune organs.

Quantitative Determination of Quercitrin Levels in Rat Plasma Using UHPLC-MS/MS and its Application in a Pharmacokinetic Study after the Oral Administration of Polygoni cuspidati Folium Capsules.

BACKGROUND: Quercitrin is widely found in herbal medicines, and it is particularly important in the design of new therapeutic agents. Because of its wide range of biological activities, methods for detecting quercitrin and its pharmacokinetics in biological samples must be investigated. OBJECTIVES: To develop and validate a sensitive and reliable ultra-high-performance liquid chromatography- tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative determination of quercitrin levels in rat plasma, and test its application in a pharmacokinetic investigation after the oral administration of Polygoni cuspidati folium capsules (HC). METHODS: First, a rapid analytical method implementing UHPLC-MS/MS for the quantification of quercitrin levels in rat plasma was developed and validated. The analyte and internal standard (IS) tinidazole were extracted from rat plasma via protein precipitation with 800 µL of methanol and 50 µL of 1% formic acid solution. Chromatographic separation was performed using an Agilent ZORBAX C18 column within 4 min. Mass spectrometry was performed for quantification using a triple-quadrupole mass spectrometer employing electrospray ionization in the negative ion mode. The MRM transitions for quercitrin and IS were m/z 447.2→229.9 and m/z 246.0→125.8, respectively. The UHPLC-MS/MS method for the quantitative determination of quercitrin levels in rat plasma was then applied to investigate its pharmacokinetics after the oral administration of HC in rats. RESULTS: The developed UHPLC-MS/MS method for detecting quercitrin in rat plasma was linear over the range of 0.1-160 ng/mL. The linear regression equation was Y = (0.7373 ± 0.0023)X - (0.0087 ± 0.0021) (r2 = 0.9978). The intra- and interday precision values were within 7.8%, and the recoveries of quercitrin and IS exceeding 67.3%. The UHPLC-MS/MS method was successfully applied to characterize the pharmacokinetic profile of quercitrin in eight rats after the oral administration of HC. The experimentally obtained values were fit to a one-compartment, first-order pharmacokinetic model, and they appeared to fit the concentration-time curve. CONCLUSION: Quercitrin was proven to be stable during sample storage, preparation, and the analytical procedures. The pharmacokinetic parameters suggested that quercitrin may be present in the peripheral tissues of rats.

Distinct and Coordinated Regulation of Small Non-coding RNAs by E2f1 and p53 During Drosophila Development and in Response to DNA Damage.

Small non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), play a pivotal role in biological processes. A comprehensive quantitative reference of small ncRNAs expression during development and in DNA damage response (DDR) would significantly advance our understanding of their roles. In this study, we systemically analyzed the expression profile of miRNAs and piRNAs in wild-type flies, e2f1 mutant, p53 mutant and e2f1 p53 double mutant during development and after X-ray irradiation. By using small RNA sequencing and bioinformatic analysis, we found that both miRNAs and piRNAs were expressed in a dynamic mode and formed 4 distinct clusters during development. Notably, the expression pattern of miRNAs and piRNAs was changed in e2f1 mutant at multiple developmental stages, while retained in p53 mutant, indicating a critical role of E2f1 played in mediating small ncRNAs expression. Moreover, we identified differentially expressed (DE) small ncRNAs in e2f1 mutant and p53 mutant after X-ray irradiation. Furthermore, we mapped the binding motif of E2f1 and p53 around the small ncRNAs. Our data suggested that E2f1 and p53 work differently yet coordinately to regulate small ncRNAs expression, and E2f1 may play a major role to regulate miRNAs during development and after X-ray irradiation. Collectively, our results provide comprehensive characterization of small ncRNAs, as well as the regulatory roles of E2f1 and p53 in small ncRNAs expression, during development and in DNA damage response, which reveal new insights into the small ncRNAs biology.

An injectable peptide hydrogel with excellent self-healing ability to continuously release salvianolic acid B for myocardial infarction.

Drug-loaded hydrogels can improve blood supply and inhibit extracellular matrix degradation after myocardial infarction. However, due to the continual dynamic motion of cardiac tissue, the hydrogel structure cannot be reconstructed in time, causing accelerated degradation and drug burst release. Here, a novel, superior, self-healing elastin-mimic peptide hydrogel (EMH) was fabricated for the local delivery of salvianolic acid B (SaB). The self-healing ability of EMH is enhanced by SaB-loaded polydopamine nanoparticles (SaB-PDA). In vitro, the pre-hydrogel (SaB-PDA/pre-EMH) is endowed with excellent biocompatibility and a low viscosity, making it suitable for intramyocardial injection. Once injected into the myocardial infarction (MI) region, SaB-PDA/pre-EMH can form SaB-PDA/EMH with great mechanical strength under the action of upregulated transglutaminase (TGase) in heart tissue post-MI. The superior self-healing ability of SaB-PDA/EMH allows for an increase in retention time in the beating ventricular wall. Therefore, with long-term release of SaB, SaB-PDA/EMH can inhibit ventricular remodeling and promote angiogenesis for MI treatment.

Drosophila Caliban preserves intestinal homeostasis and lifespan through regulating mitochondrial dynamics and redox state in enterocytes.

Precise regulation of stem cell activity is crucial for tissue homeostasis. In Drosophila, intestinal stem cells (ISCs) maintain the midgut epithelium and respond to oxidative challenges. However, the connection between intestinal homeostasis and redox signaling remains obscure. Here we find that Caliban (Clbn) functions as a regulator of mitochondrial dynamics in enterocytes (ECs) and is required for intestinal homeostasis. The clbn knock-out flies have a shortened lifespan and lose the intestinal homeostasis. Clbn is highly expressed and localizes to the outer membrane of mitochondria in ECs. Mechanically, Clbn mediates mitochondrial dynamics in ECs and removal of clbn leads to mitochondrial fragmentation, accumulation of reactive oxygen species, ECs damage, activation of JNK and JAK-STAT signaling pathways. Moreover, multiple mitochondria-related genes are differentially expressed between wild-type and clbn mutated flies by a whole-genome transcriptional profiling. Furthermore, loss of clbn promotes tumor growth in gut generated by activated Ras in intestinal progenitor cells. Our findings reveal an EC-specific function of Clbn in regulating mitochondrial dynamics, and provide new insight into the functional link among mitochondrial redox modulation, tissue homeostasis and longevity.

Thyroid receptor-interacting protein 13 and EGFR form a feedforward loop promoting glioblastoma growth.

Epidermal growth factor receptor (EGFR) amplification and EGFRvIII mutation drive glioblastoma (GBM) pathogenesis, but their regulation remains elusive. Here we characterized the EGFR/EGFRvIII "interactome" in GBM and identified thyroid receptor-interacting protein 13 (TRIP13), an AAA + ATPase, as an EGFR/EGFRvIII-associated protein independent of its ATPase activity. Functionally, TRIP13 augmented EGFR pathway activation and contributed to EGFR/EGFRvIII-driven GBM growth in GBM spheroids and orthotopic GBM xenograft models. Mechanistically, TRIP13 enhanced EGFR protein abundance in part by preventing Cbl-mediated ubiquitination and proteasomal degradation. Reciprocally, TRIP13 was phosphorylated at tyrosine(Y) 56 by EGFRvIII and EGF-activated EGFR. Abrogating TRIP13 Y56 phosphorylation dramatically attenuated TRIP13 expression-enhanced EGFR signaling and GBM cell growth. Clinically, TRIP13 expression was upregulated in GBM specimens and associated with poor patient outcome. In GBM, TRIP13 localized to cell membrane and cytoplasma and exhibited oncogenic effects in vitro and in vivo, depending on EGFR signaling but not the TRIP13 ATPase activity. Collectively, our findings uncover that TRIP13 and EGFR form a feedforward loop to potentiate EGFR signaling in GBM growth and identify a previously unrecognized ATPase activity-independent mode of action of TRIP13 in GBM biology.

A novel ß-cyclodextrin-rhein conjugate for improving the water solubility and bioavailability of rhein.

Rhein is a potential antitumor agent, but the poor water-solubility restricts its clinical applicability. ß-cyclodextrin-drug conjugates provide a possibility to improve the water-solubility of rhein and thereby enhance its bioavailability. A novel ß-cyclodextrin-rhein conjugate (ß-CD-RH) was synthesized by covalently link ß-cyclodextrin with rhein through a 1,8-diamino-3,6-dioxaoctane linker. The structure of ß-CD-RH was characterized by 1H NMR, FT-IR, Maldi-tof MS etc. The inclusion style of ß-CD-RH in water was detected by 2D NMR. The 2D ROESY spectrum provided details of the rhein moiety encapsulated in the ß-CD cavity. The water-solubility of ß-CD-RH is up to 3.24 µmol/mL ß-CD-RH exhibited higher cytotoxicity than rhein and rhein/ß-CD mixture against Hela cells. Our work provides a new way for the preparation of novel ß-CD-drug conjugate.

Structural characterization and in vitro-in vivo evaluation of effect of a polysaccharide from Sanguisorba officinalis on acute kidney injury.

We report here an acidic polysaccharide, namely RSP-3, which ameliorates acute kidney injury and is obtained from Sanguisorba officinalis. We extracted and purified two polysaccharides from this herb based on the acidity and screened them for their effect in regulating the immunological activity of macrophages. Among them, RSP-3 exhibited significant anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated macrophages by decreasing TNF-α and IL-6 levels. Subsequently, we found that RSP-3 suppressed ER stress, reduced ROS production and blocked NF-κBp65 translocation. After fully characterizing RSP-3 with a series of analytical technologies, we tested its anti-acute kidney injury (AKI) effect in vivo. In a murine AKI model induced by LPS, treatment with RSP-3 effectively ameliorated renal function. Besides, it decreased the levels of TNF-α and IL-6 in serum and reduced macrophage infiltration in injured kidney tissue. In sum, RSP-3, with a significant protective effect against AKI by showing anti-inflammatory activity, may become a meaningful drug candidate for treatment of AKI.

In Silico Design of MDM2-Targeting Peptides from a Naturally Occurring Constrained Peptide.

Naturally occurring constrained peptides are frequently used as scaffolds for bioactive peptide grating due to their high stability. Here, we used in silico methods to design several constrained peptides comprising a scorpion toxin scaffold, a MDM2 binding epitope, and a cluster of positively charged residues. The designed peptides displayed varied binding affinity to MDM2 despite differing by only one or two residues. One of the peptides, SC426, had nanomolar binding affinity (KD =6.6±2.6 nm) to MDM2, and exhibited stronger inhibitory activity on the proliferation of HCT116 cells (p53-wild type) and SW480 cells (p53-mutant) than that of nutlin-3a. Binding mode analysis of the designed peptide at MDM2 suggests that the conserved "FWL" epitope was buried in the hydrophobic binding pocket, and the residues located at the periphery of the binding site contributed to the high binding affinity of SC426. Overall, in silico design of miniproteins with therapeutic potential through epitope grafting to the naturally occurring constrained peptide is an effective strategy.

MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure.

Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here, we describe a phenomenon termed MISTERMINATE (mitochondrial-stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs, including complex-I 30kD subunit (C-I30) mRNA, occurring on the mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson's disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

Characterization of TNF-induced cell death in Drosophila reveals caspase- and JNK-dependent necrosis and its role in tumor suppression.

Tumor-necrosis factor (TNF) and its superfamily members are pleiotropic cytokines. Activation of TNF can lead to distinct cellular outcomes including inflammation, cell survival, and different forms of cell death, such as apoptosis and necrosis in a context-dependent manner. However, our understanding of what determines the versatile functions of TNF is far from complete. Here, we examined the molecular mechanisms that distinguish the forms of cell death induced by Eiger (Egr), the sole homolog of TNF in Drosophila. We show that expression of Egr in the developing Drosophila eye simultaneously induces apoptosis and apoptosis-independent developmental defects indicated by cellular disorganization, both of which rely on the c-Jun N-terminal kinase (JNK) signaling activity. Intriguingly, when effector caspases DrICE and Dcp-1 are defective or inhibited, expression of Egr triggers necrosis which is characterized by loss of cell membrane integrity, translucent cytoplasm, and aggregation of cellular organelles. Moreover, such Egr-induced necrosis depends on the catalytic activity of the initiator caspase Dronc and the input from JNK signaling but is independent of their roles in apoptosis. Further mosaic analysis with mutants of scribble (scrib), an evolutionarily conserved tumor suppressor gene regulating cell polarity, suggests that Egr/JNK-mediated apoptosis and necrosis establish a two-layered defense system to inhibit the oncogenic growth of scrib mutant cells. Together, we have identified caspase- and JNK-dependent mechanisms underlying Egr-induced apoptosis versus necrosis and their fail-safe roles in tumor suppression in an intact organism in vivo.

Identifying risk factors for high-dose methotrexate-induced toxicities in children with acute lymphoblastic leukemia.

BACKGROUND: Whether monitoring of the methotrexate (MTX) concentrations after high-dose MTX (HD-MTX) infusion can predict toxicities is still controversial, especially when HD-MTX therapy is used in the treatment of children with acute lymphoblastic leukemia (ALL), which is different than the previous schedules. The relationship between patient characteristics and severe adverse events (AEs) has yet to be determined. OBJECTIVE: To analyze the relationship between the MTX concentration and toxicities and to identify the risk predictors from patient characteristics for severe AEs during HD-MTX therapy in children with ALL. METHODS: We conducted a retrospective study on children with ALL who were treated with 388 HD-MTX infusions. The chi-square test and univariate and logistic regression analyses were used to analyze the relationship between the MTX concentrations and toxicities and to identify predictors for severe AEs. RESULTS: Febrile neutropenia (P=0.000) and vomiting (P=0.034) were more likely to occur if the infusion had an MTX level ≥1 µmol/L at 44 h, but other toxicities had no correlations with MTX concentration. Predictive factors for toxicities were as follows: higher risk stratification and higher values of albumin (ALB) for leucopenia, higher values of white blood cell count (WBC) for anemia, higher values of ALB and creatinine (Cr) for neutropenia, higher risk stratification and higher 44-h MTX concentration for febrile neutropenia, higher values of alanine transferase (ALT) for elevated ALT, higher values of ALT for elevated aspartate transferase (AST), and higher values of total bilirubin (TBil) for vomiting. CONCLUSION: Routine monitoring of 44-h MTX concentrations is essential to identify patients at high risk of developing febrile neutropenia and vomiting. This study may provide a reference for clinicians to distinguish patients with a relatively high risk of severe AEs based on certain characteristics before HD-MTX infusion.

Characterization of an Alkaline Alginate Lyase with pH-Stable and Thermo-Tolerance Property.

Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, aly08, was cloned from the marine bacterium Vibrio sp. SY01 and expressed in Escherichia coli. The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 °C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0-10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications.

Decomposition of Nickel(⠡)-Ethylenediaminetetraacetic acid by Fenton-Like reaction over oxygen vacancies-based Cu-Doped Fe3O4@γ-Al2O3 catalyst: A synergy of oxidation and adsorption.

Nickel (Ⅱ)-ethylenediaminetetraacetic acid (Ni-EDTA) complexes are widely present in electroplating effluents. Owing to its chemical stability, Ni-EDTA is hardly removed in traditional Fenton/Fenton-like processes with conventional iron (Fe)-based catalyst. In this study, oxygen vacancies were introduced into our highly efficient and novel Fe3O4@γ-Al2O3 catalysts using Cu doping for Ni-EDTA decomposition in Fenton-like system. Without noble-metal cocatalyst, the introduction of oxygen vacancies in Cu-doped Fe3O4@γ-Al2O3 catalysts exhibit excellent Fenton-like activity even in neutral or alkaline conditions. Experimental results revealed that, without the aid of extra energy, Ni-EDTA complexes could be effectively decomposed over oxygen vacancies-based catalyst. Electron paramagnetic resonance (EPR), X-ray photoelectron spectroscopy (XPS), oxygen temperature-programmed desorption (O2-TPD), and hydrogen temperature-programmed reduction (H2-TPR) were used to get a deep insight into the decomposition mechanism. Additionally, by employing the Al-containing support, stable layered double-hydroxide phases of NiAl could be formed, indicating that a synergy of oxidation and adsorption could simultaneously take place, which led to the recovery of released Ni2+ ions and also reduction in secondary pollution. To investigate the decomposition process of Ni-EDTA over oxygen vacancies-based catalyst, liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (LC-MS/MS) was employed to identify the generated intermediates, and thus, a plausible decomposition pathway was successfully conceived.

The Herb-Drug Interaction of Clopidogrel and Xuesaitong Dispersible Tablet by Modulation of the Pharmacodynamics and Liver Carboxylesterase 1A Metabolism.

OBJECTIVE: Clopidogrel and Xuesaitong dispersible tablet (XST) have been clinically proven to be effective for treating cardiocerebrovascular disease. The present study was to investigate the herb-drug interaction of Clopidogrel and XST by modulation of the pharmacodynamics and liver Carboxylesterase 1A(CES1A) metabolism. METHODS: 30 male SD rats were randomly divided into a control group (equal volumes of saline, 6 rats for mRNA analysis), a clopidogrel group (clopidogrel with dose 30 mg/kg), and a combination group (clopidogrel and XST, with dose 30 and 50 mg/kg respectively, each group continuous administration once daily for 30 days). The clopidogrel and combination group comprised 12 rats, with 6 designated for mRNA analysis and 6 for the pharmacokinetic study. The 2-bromo-3'-methoxyacetophenone- (MPB-) derivatized clopidogrel active thiol metabolite (CAMD) was measured by UHPLC-MS/MS for pharmacokinetics (n=6). The expression of CES1A mRNA was examined with real-time RT-PCR (n=6). Molecular simulation was used to investigate the inhibition effect of XST on the CES1A protein. The CAMD pharmacodynamics and CES1A metabolism were investigated to evaluated the herb-drug interaction. RESULTS: Clopidogrel and XST coadministration appreciably increased the Cmax, AUC, and MRT of CAMD. However, the expression of CES1A mRNA was decreased accordingly. It also indicated that the bioactive components in XST had good interaction with the CES1A metabolism target by molecular simulation. The animal study indicated that clopidogrel and XST coadministration produced significant herb-drug interactions at active CAMD pharmacokinetic and CES1A metabolic enzyme aspect. CONCLUSION: 30-days dose of coadministration altered hepatic CES1A protein and resulted in reduced plasma levels of active CAMD. both the decreased CES1A mRNA expression and the inhibition on the protein were due to the combination of XST, which accordingly upregulated the pharmacokinetics of plasma active CAMD.

Drosophila Caliban mediates G1-S transition and ionizing radiation induced S phase checkpoint.

Cell cycle progression is precisely regulated by diverse extrinsic and intrinsic cellular factors. Understanding the underlying mechanisms of cell cycle regulation is essential to address how normal development and tissue homeostasis are achieved. Here, we present a novel cell cycle regulator Caliban (Clbn), the Drosophila ortholog of human Serologically defined colon cancer antigen 1 (SDCCAG1) gene. We show that ionizing radiation induces expression of clbn, and over-expression of clbn blocks G1-to-S cell cycle transition in Drosophila, while flies loss of clbn have defective S phase checkpoint in response to irradiation. Mechanistically, induced expression of clbn suppressed E2F1 activity and down-regulates the DNA replication and expression of its downstream target cyclin E, a key regulator of G1-to-S transition. Meanwhile, clbn over-expression leads to upregulation of the CDK inhibitor Dacapo (Dap), and upregulated Dap is decreased when e2f1 is over-expressed. Furthermore, expression of clbn is down-regulated in cells with e2f1 over-expression or rbf1 knockdown, indicating that Clbn and E2F1 act antagonistically in mediating G1-to-S transition. Thus we provide genetic evidence that Clbn works together with E2F1 in regulating cell cycle progression, and Clbn is required for S phase cell cycle checkpoint in response to DNA damage.