Nanoparticle-directed sub-cellular localization of doxorubicin and the sensitization breast cancer cells by circumventing GST-Mediated drug resistance

Resistance to single or multiple chemotherapeutic drugs is a major complication in clinical oncology and is one of the most common treatment limitations in patients with reoccurring cancers. Nanoparticle (NP)-based drug delivery systems (DDS's) have been shown to overcome drug resistance in cancer cells mainly by avoiding the activation of efflux pumps in these cells. We demonstrate in this work that polyester-based hyperbranched dendritic-linear (HBDL)-based NPs carrying doxorubicin (Dox) can effectively overcome microsomal glutathione transferase 1 (MGST1)-mediated drug resistance in breast cancer cells. Our DDS was much more effective at considerably lower intracellular Dox concentrations (IC₅₀ 6.3 μm vs. 36.3 μm) and achieved significantly greater reductions in viability and induced higher degrees of apoptosis (31% vs. 14%) compared to the free drug in the resistant cells. Dox-loaded HBDL NPs were found to translocate across the membranes of resistant cells via active endocytic pathways and to be transported to lysosomes, mitochondria, and the endoplasmic reticulum. A significantly lower amount of Dox accumulated in these cytoplasmic compartments in resistant cells treated with free Dox. Moreover, we found that Dox-HBDL significantly decreased the expression of MGST1 and enhanced mitochondria-mediated apoptotic cell death compared to free Dox. Dox-HBDL also markedly activated the JNK pathway that contributes to the apoptosis of drug-resistant cells. These results suggest that HBDL NPs can modulate subcellular drug distribution by specific endocytic and trafficking pathways and that this results in drug delivery that alters enzyme levels and cellular signaling pathways and, most importantly, increases the induction of apoptosis. Our findings suggest that by exploiting the cell transport machinery we can optimize the polymeric vehicles for controlled drug release to overcome drug resistance combat drug resistance with much higher efficacy.     

Identification of a C-type lectin with antiviral and antibacterial activity from pacific white shrimp Litopenaeus vannamei

C-type lectins (CTLs) play crucial roles in innate immune responses in invertebrates by recognizing and eliminating microinvaders. In this study, a CTL from pacific white shrimp Litopenaeus vannamei (LvCTL3) was identified. LvCTL3 contains a single C-type lectin-like domain (CTLD), which shows similarities to those of other shrimp CTLs and has a mutated ‘EPD’ motif in Ca²⁺-binding site 2. LvCTL3 mRNA can be detected in all tested tissues and expression of LvCTL3 in gills was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges, suggesting activation responses of LvCTL3 to bacterial, virus and immune stimulant challenges. The 5′flanking regulatory region of LvCTL3 was cloned and we identified a NF-κB binding motif in the LvCTL3 promoter region. Dual-luciferase reporter assays indicated that over-expression of L. vannamei dorsal can dramatically up regulate the promoter activity of LvCTL3, suggesting that LvCTL3 expression could be regulated through NF-κB signaling pathway. As far as we know, this is the first report on signaling pathway involve in shrimp CTLs expression. The recombinant LvCTL3 protein was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified LvCTL3 can agglutinate Gram-negative microbe Vibrio alginolyticus and V. parahaemolyticus and Gram-positive bacteria Bacillus subtilis in the presence of calcium ions, but cannot agglutinate Gram-positive bacteria Streptococcus agalactiae. The agglutination activity of LvCTL3 was abolished when Ca²⁺ was chelated with EDTA, suggesting the function of LvCTL3 is Ca²⁺-dependent. In vivo challenge experiments showed that the recombinant LvCTL3 protein can significantly reduce the mortalities of V. parahemolyticus and WSSV infection, indicating LvCTL3 might play significant roles in shrimp innate immunity defense against bacterial and viral infection.     

A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice

Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.     

Age-related decline in white matter integrity in a mouse model of tauopathy: an in vivo diffusion tensor magnetic resonance imaging study

Elevated expression of human hyperphosphorylated tau is associated with neuronal loss and white matter (WM) pathology in Alzheimer's disease (AD) and related neurodegenerative disorders. Using in vivo diffusion tensor magnetic resonance imaging (DT-MRI) at 11.1 Tesla we measured age-related alterations in WM diffusion anisotropy indices in a mouse model of human tauopathy (rTg4510) and nontransgenic (nonTg) control mice at the age of 2.5, 4.5, and 8 months. Similar to previous DT-MRI studies in AD subjects, 8-month-old rTg4510 mice showed lower fractional anisotropy (FA) values in WM structures than nonTg. The low WM FA in rTg4510 mice was observed in the genu and splenium of the corpus callosum, anterior commissure, fimbria, and internal capsule and was associated with a higher radial diffusivity than nonTg. Interestingly, rTg4510 mice showed lower estimates for the mode of anisotropy than controls at 2.5 months suggesting that changes in this diffusivity metric are detectable at an early stage preceding severe tauopathy. Immunogold electron microscopy partly supports our diffusion tensor imaging findings. At the age of 4 months, rTg4510 mice show axonal tau inclusions and unmyelinated processes. At later ages (12 months and 14 months) we observed inclusions in myelin sheath, axons, and unmyelinated processes, and a “disorganized” pattern of myelinated fiber arrangement with enlarged inter-axonal spaces in rTg4510 but not in nonTg mice. Our data support a role for the progression of tau pathology in reduced WM integrity measured by DT-MRI. Further in vivo DT-MRI studies in the rTg4510 mouse should help better discern the detailed mechanisms of reduced FA and anisotropy mode, and the specific role of tau during neurodegeneration.     

Upregulated expression of human alpha-defensins 1, 2 and 3 in hypercholesteremia and its relationship with serum lipid levels

Human alpha-defensins are natural antimicrobial peptides of neutrophils evolved in host defense reactions and circulating nonstressed alpha-defensins may be associated with serum lipid levels. The aim of this work was to examine whether the expression of alpha-defensins 1, 2 and 3 genes are changed and whether this changes are reversed following treatment in patients with hypercholesteremia. A total of 40 individuals of hypercholesteremia group were studied, compared with 40 individuals of normal control group. Protein levels and gene expression levels of alpha-defensins 1, 2 and 3 were significantly higher in patients with hypercholesteremia compared with subjects in normal control group. In patients with hypercholesteremia, protein levels of alpha-defensins 1, 2 and 3 correlated positively with the levels of total cholesterol and low-density lipoprotein cholesterol. Protein levels and gene expression levels of alpha-defensins 1, 2 and 3 were decreased significantly after a treatment with atorvastatin calcium 20 mg daily compared with the patients before the treatment. Our results suggest that the expression of alpha-defensins 1, 2 and 3 genes is involved in dyslipidemia in patients with hypercholesteremia.     

Formyl Peptide Receptor Suppresses Melanoma Development and Promotes NK Cell Migration

In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop antitumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. In our current study, wild-type mice were inoculated with melanoma cell line B16-F10 (1?×?10⁶/mouse) and treated with the formyl peptide receptor (FPR) agonist WKYMVm or the FPR antagonist WRW⁴. Growth of melanoma cell line B16-F10 was significantly inhibited in WKYMVm-treated mice and markedly promoted in WRW⁴-treated mice compared with control. Decreased number of myeloid-derived suppressor cells (MDSCs) and increased NK cell infiltration in tumor tissues were detected from WKYMVm-treated mice. Next, we showed that depletion of NK cell significantly increased tumor development in B16 tumor-bearing mice compared with the control group, and the suppressed tumor-developing effect of WKYMVm in B16 melanoma was abrogated with NK cell depletion. We also found that WKYMVm stimulates chemotactic migration in NK cells via the FPR family, and this was dependent on extracellular signal-related kinase (ERK) activation. Moreover, in our further experiment, we showed that the increased infiltration of NK cell and promoted NK cell chemotaxis in B16 melanoma induced by WKYMVm were both abolished with ERK inhibitor PD98059 administration. In conclusion, the FPR family promoted NK cell migration through ERK activation and inhibited B16 melanoma growth in a murine model.     

Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize

Genetic imprinting is a specific epigenetic phenomenon in which a subset of genes is expressed depending on their parent-of-origin. Two types of chromatin modifications, DNA methylation and histone modification, are generally believed to be involved in the regulation of imprinting. However, the genome-wide correlation between allele-specific chromatin modifications and imprinted gene expression in maize remains elusive. Here we report genome-wide high resolution allele-specific maps of DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) in maize endosperm. For DNA methylation, thousands of parent-of-origin dependent differentially methylated regions (pDMRs) were identified. All pDMRs were uniformly paternally hypermethylated and maternally hypomethylated. We also identified 1131 allele-specific H3K27me3 peaks that are preferentially present in the maternal alleles. Maternally expressed imprinted genes (MEGs) and paternally expressed imprinted genes (PEGs) had different patterns of allele-specific DNA methylation and H3K27me3. Allele-specific expression of MEGs was not directly related to allele-specific H3K27me3, and only a subset of MEGs was associated with maternal-specific DNA demethylation, which was primarily located in the upstream and 5′ portion of gene body regions. In contrast, allele-specific expression of a majority of PEGs was related to maternal-specific H3K27me3, with a subgroup of PEGs also associated with maternal-specific DNA demethylation. Both pDMRs and maternal H3K27me3 peaks associated with PEGs are enriched in gene body regions. Our results indicate highly complex patterns of regulation on genetic imprinting in maize endosperm.Genetic imprinting is an epigenetic phenomenon, where genes are expressed in a parent-of-origin dependent manner in many plant species and mammals. Although first discovered in plants (Kermicle and Alleman 1990), research on genetic imprinting is much more advanced in mammals in terms of the number of imprinted genes identified and the understanding of their regulatory mechanisms (Koerner and Barlow 2010; Barlow 2011; Bartolomei and Ferguson-Smith 2011). Only a small number of imprinted genes were observed in plants for a long time since the phenomenon is highly specific to the triploid endosperm (Raissig et al. 2011). However, recent studies have indicated that genetic imprinting in plants is much more prevalent than previously thought, with hundreds of genes shown to be imprinted in several plant species (Gehring et al. 2011; Hsieh et al. 2011; Luo et al. 2011; Waters et al. 2011; Zhang et al. 2011). In contrast to mammals, where the regulation of genetic imprinting has been extensively studied (Koerner and Barlow 2010; Barlow 2011; Abramowitz and Bartolomei 2012), the understanding of regulation of parental imprinting in plants is highly limited.DNA methylation is one of the primary modifications reported to be associated with genetic imprinting. In Arabidopsis, DNA methylation around several maternally expressed imprinted protein-coding genes (MEG) including FWA, FIS2, and MPC was shown to be important for their maternally preferred expression, as all these genes exhibited biallelic expression in endosperm fertilized with met1 pollen (Kinoshita et al. 2004; Jullien et al. 2006; Tiwari et al. 2008). Two studies using RNA-seq in Arabidopsis also showed that a number of MEGs exhibited biallelic expression in paternal met1 endosperm, and the maternal alleles of dozens of paternally expressed imprinted genes (PEGs) were reactivated in maternal dme endosperm (Hsieh et al. 2011; Wolff et al. 2011). In maize, five confirmed endosperm MEGs (Fie1, Fie2, Mez1, Meg1, and Mee1) contain differentially methylated regions (DMRs) (Gutierrez-Marcos et al. 2004; Gutierrez-Marcos et al. 2006; Haun et al. 2007), and activation of the Fie1 maternal allele in the endosperm requires DNA demethylation of the maternal allele (Hermon et al. 2007).Another modification associated with genetic imprinting involves histone methylation. Polycomb repressive complex 2 (PRC2) is known to mediate the trimethylation of histone H3 lysine 27 (H3K27me3) (Schuettengruber and Cavalli 2009). Results on PHE1, the only well-studied PEG in plants, indicated that silencing of its maternal allele depends on a functional PRC2 complex in addition to DNA demethylation (Kohler et al. 2003, 2005; Makarevich et al. 2008). Recently, a number of MEGs and PEGs were shown to be biallelically expressed in maternal fie or fis2 endosperm (Hsieh et al. 2011; Wolff et al. 2011).Although the studies above suggest that DNA methylation and the PRC2 complex could be responsible for monoallelic expression of imprinted genes, there is not yet any general rule for the function of DNA and histone methylation on the regulation of genetic imprinting in plants. A high resolution genome-wide map of allele-specific DNA methylation and allele-specific histone modification will be crucial to gain better understanding of the regulation of genetic imprinting. Recently, several genome-wide studies have provided evidence that allele-specific patterns of DNA methylation or parent-of-origin dependent differentially methylated regions (pDMRs) are associated with some imprinted genes in mice and plants (Zhang et al. 2011; Ibarra et al. 2012; Xie et al. 2012; Rodrigues et al. 2013). Several studies in mammals also suggest a mutually exclusive relationship between allele-specific DNA methylation and histone modification or among different histone modifications (Xin et al. 2001; Fournier et al. 2002; Carr et al. 2007; Lindroth et al. 2008; Singh et al. 2010; Hon et al. 2012).Here we report a genome-wide analysis of allele-specific DMRs, H3K27me3, and imprinted gene expression in maize endosperm. Thousands of pDMRs and allele-specific H3K27me3 peaks were identified. Correlation of pDMRs, allele-specific H3K27me3 profile, and the expression of imprinted genes showed that MEGs and PEGs have different patterns of DNA methylation and H3K27me3. This study reveals complex patterns of genetic imprinting regulation in maize endosperm.