Interspecific root interaction enhances cadmium accumulation in Oryza sativa when intercropping with cadmium accumulator Artemisia argyi.

The contamination of arable land with heavy metals, such as Cd, is a serious concern worldwide. Intercropping with Cd accumulators can be used for efficient safe crop production and phytoremediation of Cd-contaminated soil. However, the effect of intercropping on Cd uptake by main crops and accumulators varies among plant combinations. Rhizosphere interaction may mediate Cd uptake by intercropped plants, but the mechanism is unclear. Thus, in the present study, we aimed to examine the effect of rhizosphere interaction on Cd uptake by intercropping rice (Oryza sativa L.) with mugwort (Artemisia argyi Levl. et Vant.) in Cd-contaminated paddy soil. We grew O. sativa and A. argyi in pots designed to allow different levels of interaction: complete root interaction (no barrier), partial root interaction (nylon mesh barrier), and no root interaction (plastic film barrier). Our results indicated that both complete and partial root interaction increased the shoot and root mass of A. argyi, but did not decrease the shoot, root, and grain mass of O. sativa. Interspecific root interaction significantly increased the Cd content in the shoots, roots, and grains of O. sativa and the shoots of A. argyi. Increased content of total organic acids in the rhizosphere, which increased the content of available Cd, was a possible mechanism of increased Cd uptake in both plants under interspecific root interaction. Our findings demonstrate that an intercropping system can extract more Cd from contaminated soil than a monocropping system of either A. argyi or O. sativa. However, the intercropping system did not facilitate safe crop production because it substantially increased grain Cd content in O. sativa.

Impairment of antigen-presenting function of peripheral γδ T cells in patients with sepsis.

Impairment of antigen-presenting functions is a key mechanism contributing to sepsis-induced immunosuppression. Recently, γδ T cells have been demonstrated as professional antigen-presenting cells (APCs); however, their role in sepsis remains unknown. In this in vitro study, the APC function of human peripheral γδ T cells was assessed using samples collected from 42 patients with sepsis and 27 age-matched healthy controls. The APC-related markers HLA-DR, CD27, CD80, and CCR7 on fresh γδT cells were significantly higher in patients with sepsis compared with matched controls; however, they responded poorly to 4-hydroxy-3-methyl-2-butenyl pyrophosphate (HMBPP) stimulation, characterized by the deactivation of these APC markers and impaired proliferation. Furthermore, the adhesion function of γδ T cells, essential for antigen presentation, was greatly reduced in patients with sepsis; for instance, in co-cultures with green fluorescent protein-expressing Escherichia coli, HMBPP-activated γδT cells from healthy individuals adhered to E. coli efficiently, whereas no such phenomenon was observed with respect to γδT cells from patients with sepsis. In line with these results, in co-cultures with isolated CD4+ αß T cells, HMBPP-activated γδT cells of healthy individuals promoted the efficient proliferation of CD4+ αß T cells, whereas γδT cells from patients with sepsis did not do so. In conclusion, our findings show that the antigen-presenting function of γδT cells is severely impaired in patients with sepsis and the mechanisms behind need further study.

Ex vivo pulsed dendritic cell vaccination against cancer.

As the most powerful antigen-presenting cell type, dendritic cells (DCs) can induce potent antigen-specific immune responses in vivo, hence becoming optimal cell population for vaccination purposes. DCs can be derived ex vivo in quantity and manipulated extensively to be endowed with adequate immune-stimulating capacity. After pulsing with cancer antigens in various ways, the matured DCs are administrated back into the patient. DCs home to lymphoid organs to present antigens to and activate specific lymphocytes that react to a given cancer. Ex vivo pulsed DC vaccines have been vigorously investigated for decades, registering encouraging results in relevant immunotherapeutic clinical trials, while facing some solid challenges. With more details in DC biology understood, new theory proposed, and novel technology introduced (featuring recently emerged mRNA vaccine technology), it is becoming increasingly likely that ex vivo pulsed DC vaccine will fulfill its potential in cancer immunotherapy.

[Study on Autophagy and Apoptosis Induced by Amiodarone Combined with Glycyrrhetinic Acid in HepG2 Cells].

OBJECTIVE: To investigate the effects of amiodarone combined with glycyrrhetinic acid on the activity, apoptosis and autophagy in human hepatoma HepG2 cells. METHODS: After using amiodarone and glycyrrhetinic acid alone or in combination treatment for HepG2 cells, MTT assay was used to detect cell proliferation, Annexin Ⅴ/PI flow cytometry was used to detect apoptosis; Western blot was used to detect the expression of autophagy-related proteins Beclin-1, LC3 and p62. The formation of EGFP-LC3 green fluorescent aggregates was observed under a fluorescence microscope. The effects of autophagy on cell proliferation and apoptosis were studied by autophagy inhibitor hydroxychloroquine (HCQ) and autophagy promoter Rapamycin. RESULTS: The cell viability in combination group was lower than that in single drug group, and the apoptosis rate was higher than that in single drug group. Compared with single-drug group, the expressions of Beclin-1 and LC3Ⅱ protein in the combination group were higher than that in the single-drug group, while the expression of p62 protein was lower in the single-drug group. Fluorescence microscopy results showed that the number of EGFP-LC3 fluorescent aggregates in the combination group were more than that in the single-drug. Using amiodarone and glycyrrhetinic acid treated HepG2 cells, inhibition of auotophagy could decrease cell viability, increase apoptosis rate of cells; promoting autophagy would decrease the apoptosis rate and increase cells viability. CONCLUSION: By increasing apoptosis of hepatocellular carcinoma HepG2 cells and autophagy level, and decreasing the cell activity, amiodarone combining with glycyrrhetinic acid treatment inducing autophagy a protective mechanism for cells.

Correction: Yan Chen, et al. Dual Agent Loaded PLGA Nanoparticles Enhanced Antitumor Activity in a Multidrug-Resistant Breast Tumor Eenograft Model. Int. J. Mol. Sci. 2014, 15, 2761-2772.

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Dual agent loaded PLGA nanoparticles enhanced antitumor activity in a multidrug-resistant breast tumor eenograft model.

Multidrug-resistant breast cancers have limited and ineffective clinical treatment options. This study aimed to develop PLGA nanoparticles containing a synergistic combination of vincristine and verapamil to achieve less toxicity and enhanced efficacy on multidrug-resistant breast cancers. The 1:250 molar ratio of VCR/VRP showed strong synergism with the reversal index of approximately 130 in the multidrug-resistant MCF-7/ADR cells compared to drug-sensitive MCF-7 cells. The lyophilized nanoparticles could get dispersed quickly with the similar size distribution, zeta potential and encapsulation efficiency to the pre-lyophilized nanoparticles suspension, and maintain the synergistic in vitro release ratio of drugs. The co-encapsulated nanoparticle formulation had lower toxicity than free vincristine/verapamil combinations according to the acute-toxicity test. Furthermore, the most effective tumor growth inhibition in the MCF-7/ADR human breast tumor xenograft was observed in the co-delivery nanoparticle formulation group in comparison with saline control, free vincristine, free vincristine/verapamil combinations and single-drug nanoparticle combinations. All the data demonstrated that PLGANPs simultaneously loaded with chemotherapeutic drug and chemosensitizer might be one of the most potential formulations in the treatment of multidrug-resistant breast cancer in clinic.

Development of lipid-shell and polymer core nanoparticles with water-soluble salidroside for anti-cancer therapy.

Salidroside (Sal) is a potent antitumor drug with high water-solubility. The clinic application of Sal in cancer therapy has been significantly restricted by poor oral absorption and low tumor cell uptake. To solve this problem, lipid-shell and polymer-core nanoparticles (Sal-LPNPs) loaded with Sal were developed by a double emulsification method. The processing parameters including the polymer types, organic phase, PVA types and amount were systemically investigated. The obtained optimal Sal-LPNPs, composed of PLGA-PEG-PLGA triblock copolymers and lipids, had high entrapment efficiency (65%), submicron size (150 nm) and negatively charged surface (-23 mV). DSC analysis demonstrated the successful encapsulation of Sal into LPNPs. The core-shell structure of Sal-LPNPs was verified by TEM. Sal released slowly from the LPNPs without apparent burst release. MTT assay revealed that 4T1 and PANC-1 cancer cell lines were sensitive to Sal treatment. Sal-LPNPs had significantly higher antitumor activities than free Sal in 4T1 and PANC-1 cells. The data indicate that LPNPs are a promising Sal vehicle for anti-cancer therapy and worthy of further investigation.

α, ω-Cholesterol-functionalized low molecular weight polyethylene glycol as a novel modifier of cationic liposomes for gene delivery.

Here, three novel cholesterol (Ch)/low molecular weight polyethylene glycol (PEG) conjugates, termed α, ω-cholesterol-functionalized PEG (Ch2-PEGn), were successfully synthesized using three kinds of PEG with different average molecular weight (PEG600, PEG1000 and PEG2000). The purpose of the study was to investigate the potential application of novel cationic liposomes (Ch2-PEGn-CLs) containing Ch2-PEGn in gene delivery. The introduction of Ch2-PEGn affected both the particle size and zeta potential of cationic liposomes. Ch2-PEG2000 effectively compressed liposomal particles and Ch2-PEG2000-CLs were of the smallest size. Ch2-PEG1000 and Ch2-PEG2000 significantly decreased zeta potentials of Ch2-PEGn-CLs, while Ch2-PEG600 did not alter the zeta potential due to the short PEG chain. Moreover, the in vitro gene transfection efficiencies mediated by different Ch2-PEGn-CLs also differed, in which Ch2-PEG600-CLs achieved the strongest GFP expression than Ch2-PEG1000-CLs and Ch2-PEG2000-CLs in SKOV-3 cells. The gene delivery efficacy of Ch2-PEGn-CLs was further examined by addition of a targeting moiety (folate ligand) in both folate-receptor (FR) overexpressing SKOV-3 cells and A549 cells with low expression of FR. For Ch2-PEG1000-CLs and Ch2-PEG2000-CLs, higher molar ratios of folate ligand resulted in enhanced transfection efficacies, but Ch2-PEG600-CLs had no similar in contrast. Additionally, MTT assay proved the reduced cytotoxicities of cationic liposomes after modification by Ch2-PEGn. These findings provide important insights into the effects of Ch2-PEGn on cationic liposomes for delivering genes, which would be beneficial for the development of Ch2-PEGn-CLs-based gene delivery system.

Study on biopharmaceutics classification and oral bioavailability of a novel multikinase inhibitor NCE for cancer therapy.

Specific biopharmaceutics classification investigation and study on phamacokinetic profile of a novel drug candidate (2-methylcarbamoyl-4-{4-[3- (trifluoromethyl) benzamido] phenoxy} pyridinium 4-methylbenzenesulfonate monohydrate, NCE) were carried out. Equilibrium solubility and intrinsic dissolution rate (IDR) of NCE were estimated in different phosphate buffers. Effective intestinal permeability (P(eff)) of NCE was determined using single-pass intestinal perfusion technique in rat duodenum, jejunum and ileum at three concentrations. Theophylline (high permeability) and ranitidine (low permeability) were also applied to access the permeability of NCE as reference compounds. The bioavailability after intragastrical and intravenous administration was measured in beagle dogs. The solubility of NCE in tested phosphate buffers was quite low with the maximum solubility of 81.73 µg/mL at pH 1.0. The intrinsic dissolution ratio of NCE was 1 × 10⁻4 mg·min⁻¹·cm⁻². The P(eff) value of NCE in all intestinal segments was more proximate to the high-permeability reference theophylline. Therefore, NCE was classified as class II drug according to Biopharmaceutics Classification System due to its low solubility and high intestinal permeability. In addition, concentration-dependent permeability was not observed in all the segments, indicating that there might be passive transportation for NCE. The absolute oral bioavailability of NCE in beagle dogs was 26.75%. Therefore, dissolution promotion will be crucial for oral formulation development and intravenous administration route will also be suggested for further NCE formulation development. All the data would provide a reference for biopharmaceutics classification research of other novel drug candidates.

A novel fluoropolymer as a protein delivery vector with robust adjuvant effect for cancer immunotherapy.

The inefficient treatment using protein-based nanovaccines is largely attributed to their inadequate immunogenicity. Herein, we developed a novel fluoropolymer (PF) via ring-opening polymerization and constructed a fluoropolymer-based nanovaccine for tumor immunotherapy. Due to the existence of fluoroalkyl chains, PF not only played a crucial role in tumor antigen delivery but also exhibited a remarkable adjuvant effect in enhancing the immunogenicity of nanovaccines. The nanovaccines formed by mixing PF with a model antigen ovalbumin (OVA) enhanced the uptake of antigen proteins by dendritic cells (DCs) and promoted the maturation and antigen presentation of DCs. Compared with free OVA, PF/OVA showed better efficacy in both pre-cancer prevention and tumor treatment. Furthermore, the proportion of CD4+ T and CD8+ T cells was significantly increased in lymph nodes and tumors of mice immunized with PF/OVA. Additionally, there was a great enhancement in the levels of key anti-tumor cytokines (TNF-α and IFN-γ) in the serum of the PF/OVA immunized mice. Our research has shown that fluoropolymer PF applied as a protein vector and adjuvant has great potential for the development of nanovaccines with robust immunogenicity.

Growth-inhibitory and apoptosis-inducing effects of tanshinones on hematological malignancy cells and their structure-activity relationship.

This study has investigated the growth-inhibitory and apoptosis-inducing effects of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone on hematological malignancy cell lines, aiming to explore their structure-activity relationship. The growth-inhibitory effects of the tanshinones on K562 and Raji cells were assessed using a modified MTT assay; the apoptosis-inducing effects were assessed by fluorescence microscopy and flow cytometry analysis. The changes in cellular morphology were observed using an inverted phase-contrast microscope. MTT results revealed that these tanshinones inhibited cell proliferation in a concentration-dependent and time-dependent manner. The IC50 values of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone for K562 cells were 3.50, 13.52, 19.32, and 47.52 µmol/l at 24 h; 1.36, 4.70, 5.67, and 22.72 µmol/l at 48 h; and 1.15, 1.59, 2.82, and 19.53 µmol/l at 72 h, and the values for Raji cells were 3.30, 4.37, 12.92, and 52.36 µmol/l at 24 h; 1.55, 1.71, 6.54, and 25.45 µmol/l at 48 h; and 1.07, 1.38, 1.89, and 18.47 µmol/l at 72 h. The flow cytometry analysis demonstrated that these tanshinones induced apoptosis of K562 cells in a concentration-dependent manner, and dihydrotanshinone as well as tanshinone I were more potent than tanshinone IIA and cryptotanshinone. Some noticeable apoptotic morphologies could be observed by fluorescence microscopy on tanshinones-treated Raji cells. Collectively, these tanshinones caused growth inhibition and apoptosis in hematological malignancy cell lines, with dihydrotanshinone being the most potent, followed by tanshinone I, tanshinone IIA, and cryptotanshinone. These results suggested that the structure of aromatic ring A enhanced the cytotoxicity and the structure of ring C may have contributed to the cytotoxicity, but the mechanisms need to be further investigated.

Ecological drivers of avian diversity in a subtropical landscape: Effects of habitat diversity, primary productivity and anthropogenic disturbance.

Understanding the roles of ecological drivers in shaping biodiversity is fundamental for conservation practice. In this study, we explored the effects of elevation, conservation status, primary productivity, habitat diversity and anthropogenic disturbance (represented by human population density and birding history) on taxonomic, phylogenetic and functional avian diversity in a subtropical landscape in southeastern China. We conducted bird surveys using 1-km transects across a total of 30 sites, of which 10 sites were located within a natural reserve. Metrics of functional diversity were calculated based on six functional traits (body mass, clutch size, dispersal ratio, sociality, diet and foraging stratum). We built simultaneous autoregression models to assess the association between the ecological factors and diversity of the local avian communities. Local avian diversity generally increased with increasing habitat diversity, human population density and primary productivity. We also detected phylogenetic and functional clustering in these communities, suggesting that the avian assemblages were structured mainly by environmental filtering, rather than interspecific competition. Compared with sites outside the natural reserve, sites within the natural reserve had relatively lower avian diversity but a higher level of phylogenetic heterogeneity.

Telmisartan Attenuates the Growth of Epithelium-like Cells and Glomerular Injury in Spontaneously Hypertensive Rats.

The abnormal growth of epithelium-like cells has been noticed in spontaneously hypertensive rats (SHRs) with hypertensive nephropathy. However, the characteristics of abnormal epithelium-like cells and their pathogenesis in hypertensive nephropathy are not fully understood. In the present study, we investigated the correlation of epithelium-like cells with glomerular injury, and the effects of early drug intervention with telmisartan, an anti-hypertensive drug, on the growth of epithelium-like cells. The results showed that the epithelium-like cells were obviously observed lining along the luminal surface of Bowman's capsule in glomeruli, significantly resulting in the atrophy of the glomerular tuft. Some of the epithelium-like cells strongly expressed proliferating cell nuclear antigen (PCNA) and vimentin, indicating active cellular proliferation. The incidence of epithelium-like cells varied from 13.6% to 54.4% of glomeruli in 48-week-old SHRs, and from 5.1% to 18.0% of glomeruli in age-matched Wistar-Kyoto (WKY) rats (P<0.01). The linear regression analysis further confirmed an obvious correlation between the incidence of epithelium-like cells and the glomerular injury. Moreover, early intervention with telmisartan could dramatically attenuate the progression of epithelium-like cells growth. However, no significant effect of telmisartan on the established epithelium-like cells was observed. Taken together, we demonstrated the involvement of abnormal epithelium-like cells growth in glomerular injury during hypertensive nephropathy in SHRs, and firstly showed the positive effects of the anti-hypertensive drug on the progression of epithelium-like cells growth.

Novel thienopyridine derivatives as specific anti-hepatocellular carcinoma (HCC) agents: synthesis, preliminary structure-activity relationships, and in vitro biological evaluation.

Novel thienopyridine derivatives 1b-1r were synthesized, based on a hit compound 1a that was found in a previous cell-based screening of anticancer drugs. Compounds 1a-1r have the following features: (1) their anticancer activity in vitro was first reported by our group. (2) The most potent analog 1g possesses hepatocellular carcinoma (HCC)-specific anticancer activity. It can specifically inhibit the proliferation of the human hepatoma HepG2 cells with an IC(50) value of 0.016µM (compared with doxorubicin as a positive control, whose IC(50) was 0.37µM). It is inactive toward a panel of five different types of human cancer cell lines. (3) Compound 1g remarkably induces G(0)/G(1) arrest and apoptosis in HepG2 cells in vitro at low micromolar concentrations. These results, especially the HCC-specific anticancer activity of 1g, suggest their potential in targeted chemotherapy for HCC.

[Brain targeting of gastrodin nasal in situ gel].

OBJECTIVE: To investigate the biodistribution of gastrodin ion-activated nasal in situ gel in rat blood and brain tissues and to evaluate its brain targeting. METHODS: Intravenous administration of gastrodin solution or intranasal administration of gastrodin nasal in situ gel were given to 32 rats, respectively. The concentrations of gastrodin in the plasma and gastrodigenin in the brain tissues of the rats were determined by HPLC. RESULTS: The intranasal administration of the in situ gel of gastrodin produced more significant brain targeting effect than the intravencus administration of gstrodin solution (P < 0.01). The area under curve (AUC) of cerebrum, cerebellum and olfactory bulb increased by 1.16, 0.77 and 3.34 times, with 2.66. 2.18 and 5.34 brain targeting indexes (BTI), respectively. The mean residence time (MRT) increased by nearly four-folds. CONCLUSION: Gastrodin nasal in situ gel can improve the brain targeting of gastrodin and slow its release.

[Study on drug release of gastrodin ion-activated nasal in situ gel in vitro].

OBJECTIVE: To study on the drug release characteristics and mechanism of gastrodin ion-activated nasal in situ gel in vitro. METHOD: Regularity and mechanism of the drug release of gastrodin nasal in situ gel were studied by using the diffusion cell model and the membrane-less dissolution model, respectively. A novel kinesis diffusion cell model was designed according to the characteristics of release environment of nasal cavity. It was used to investigate the effect of adhesiveness on the release of the in situ gel. RESULT: Drug release of gastrodin nasal in situ gel followed the one order release model. Erosion rate of the gel was low and not linearly correlated with the release rate. Compared with gastrodin solution, the nasal in situ gel could increase release time and release amount. CONCLUSION: Gastrodin in the nasal in situ gel is released mainly by diffusion rather than erosion. Release amount of the in situ gel in nasal cavity may be obviously increased because of its adhesiveness.

In Vivo Ovarian Cancer Gene Therapy Using CRISPR-Cas9.

Clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9) genome editing technology holds great promise for the field of human gene therapy. However, a lack of safe and effective delivery systems restricts its biomedical application. Here, a folate receptor-targeted liposome (F-LP) was used to deliver CRISPR plasmid DNA co-expressing Cas9 and single-guide RNA targeting the ovarian cancer-related DNA methyltransferase 1 (DNMT1) gene (gDNMT1). F-LP efficiently bound the gDNMT1 plasmid and formed a stable complex (F-LP/gDNMT1) that was safe for injection. F-LP/gDNMT1 effectively mutated endogenous DNMT1 in vitro, and then expressed the Cas9 endonuclease and downregulated DNMT1 in vivo. The tumor growth of both paclitaxel-sensitive and -resistant ovarian cancers were inhibited by F-LP/gDNMT1, which shows fewer adverse effects than paclitaxel injection. Therefore, CRISPR-Cas9-targeted DNMT1 manipulation may be a potential therapeutic regimen for ovarian cancer, and lipid-mediated delivery systems represent promising delivery vectors of CRISPR-Cas9 technology for precise genome editing therapeutics.

[Tumor cell targetability of folate receptor-mediated mitoxantrone albumin nanoparticles].

OBJECTIVE: To study the tumor cell targetability of folate-conjugated mitoxantone-loaded albumin nanoparticles (MTO-BSANP-folate). METHODS: Bovine albumin nanoparticles were prepared by desolvation method. The activated folic acid (N-hydroxysuccinimide ester of folic acid) was conjuated to the surface of BSANP via the amino groups. The MTO-BSANP-folate was prepared by mixing folate-conjugated albumin nanoparticles with mitoxantrone and then cross-linked by glutaraldehyde. 3HTdR and flow cytometry were used to evaluate the targetability of MTO-BSANP-folate. RESULTS: The encapsulation rate of folate-conjugated mitoxantrone albumin nanoparticles was (96.55 +/- 0.96)% and the drug loading was (9.66 +/- 0.10)%. The results of 3HTdR showed that the efficacy of MTO-BSANP-folate in killing SKOV3 cells was higher than that of MTO-BSANP-folate, and the results of flow cytometry showed that the apoptosis-promoting effect of MTO-BSANP-folate was 3.5-4.5 times higher than that of MTO-BSANP. CONCLUSION: MTO-BSANP-folate could be targeted, via folate receptor, to the tumor cells rich in folate receptors.

[Research progress in the reversion of traditional Chinese medicine on multidrug resistance of tumor].

Researches on MDR (multidrug resistance) of tumor presently focus on seeking chemosensitizers with more targets, high efficiency and low toxicity from traditional Chinese medicine. This paper reviews the research progress in the reversion of MDR of leukemia, hepatocarcinoma, breast carcinoma and oral epithelioid neoplasia by TDM compound, its extracts, its groups of active ingredients or its active ingredients.