Inhibition of coxsackievirus B3 in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosome entry site

Coxsackievirus B3 (CVB3) is a primary cause of viral myocarditis, yet no effective therapeutic against CVB3 is available. Nucleic acid-based interventional strategies against various viruses, including CVB3, have shown promise experimentally, but limited stability and inefficient delivery in vivo remain as obstacles to their potential as therapeutics. We employed phosphorodiamidate morpholino oligomers (PMO) conjugated to a cell-penetrating arginine-rich peptide, P007 (to form PPMO), to address these issues. Eight CVB3-specific PPMO were evaluated with HeLa cells and HL-1 cardiomyocytes in culture and in a murine infection model. One of the PPMO (PPMO-6), designed to target a sequence in the 3' portion of the CVB3 internal ribosomal entry site, was found to be especially potent against CVB3. Treatment of cells with PPMO-6 prior to CVB3 infection produced an approximately 3-log(10) decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when PPMO-6 treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of PPMO-6 once prior to and once after CVB3 infection showed an approximately 2-log(10)-decreased viral titer in the myocardium at 7 days postinfection and a significantly decreased level of cardiac tissue damage, compared to the controls. Thus, PPMO-6 provided potent inhibition of CVB3 amplification both in cell cultures and in vivo and appears worthy of further evaluation as a candidate for clinical development.     

Expression of chemokine-like factor 1 is upregulated during T lymphocyte activation

Chemokine-like factor 1 (CKLF1) is a cytokine with chemotactic effects on leukocytes and a functional ligand of CCR4. This cytokine is widely expressed and the level of expression is reported to be upregulated in asthma and rheumatoid arthritis (RA), disease conditions in which T lymphocytes are over-activated. In order to determine the expression profile of CKLF1 in activated T lymphocytes, we first employed a PCR-based method on human blood fractions cDNA panels and found that CKLF1 was upregulated in activated CD4+ and CD8+ cells, with no obvious changes in CD19+ cells. We further performed kinetic analyses of CKLF1 expression in phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) at both the mRNA and protein levels. In resting PBL, the constitutive expression of CKLF1 was low at mRNA level and barely detectable at the protein level; however, both were remarkably upregulated by PHA, appearing at 8h after PHA-stimulation and persisting up to 72h. These results suggest that CKLF1 may be involved in T lymphocyte activation and further study of CKLF1 function will prove valuable.     

激活蛋白处理水稻引发基因差异表达的研究

利用抑制性消减杂交技术(Suppression Subtractive Hybridization,SSH)成功构建了植物激活蛋白处理水稻与非处理组水稻中差异表达的消减cDNA文库。从文库中一共筛选到1756个克隆,通过反向Northern杂交,从中得到264个有效克隆并测序,利用BLAST在GenBank数据库进行序列相似性比对分析,获得28个上升表达差异基因。通过分析这些基因与植物的光合作用、营养物质的运输代谢等多种植物的生理生化功能相关。本研究为揭示激活蛋白的作用机理奠定基础。     

不同转移潜能肿瘤细胞肝素酶的表达

目的研究肝素酶(Heparanase,Hpa)表达水平与人类肿瘤转移的相关性。方法利用半定量RT-PCR、免疫组织化学(S-P法)和Westernblot检测2组4种不同转移潜能的人类肿瘤细胞系中HpamRNA和蛋白的表达水平。结果HpamRNA和蛋白相对表达量在高转移潜能人类肺癌细胞(0·757±0·033,0·670±0·020)、乳腺癌细胞(0·617±0·024,0·661±0·013)中明显高于相应的低转移潜能肺癌细胞(0·518±0·012,0·406±0·012)、乳腺癌细胞(0·170±0·016,0·227±0·011)。结论在所研究的人类肿瘤中,HpamRNA和蛋白的表达水平与肿瘤的转移能力呈正相关。     

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners,Hierarchy and Physiological Significance

Background

While protease-activated-receptor 1 (PAR₁) plays a central role in tumor progression, little is known about the cell signaling involved.

Methodology/Principal Findings

We show here the impact of PAR₁ cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR₁ activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR₁ C-tail did not prevent Shc-PAR₁ association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR₁ C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR₁-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR₁ -C-tail, hPar1 oncogenic properties are abrogated.

Conclusions/Significance

This is the first demonstration that a cytoplasmic portion of the PAR₁ C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR₁ -associating region in the breast cancer signaling niche.     

Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.     

Surface-Based Analysis on Shape and Fractional Anisotropy of White Matter Tracts in Alzheimer's Disease

Background

White matter disruption has been suggested as one of anatomical features associated with Alzheimer''s disease (AD). Diffusion tensor imaging (DTI), which has been widely used in AD studies, obtains new insights into the white matter structure.

Methods

We introduced surface-based geometric models of the deep white matter tracts extracted from DTI, allowing the characterization of their shape variations relative to an atlas as well as fractional anisotropy (FA) variations on the atlas surface through large deformation diffeomorphic metric mapping (LDDMM). We applied it to assess local shapes and FA variations of twenty-three deep white matter tracts in 13 patients with AD and 19 healthy control subjects.

Results

Our results showed regionally-specific shape abnormalities and FA reduction in the cingulum tract and the sagittal stratum tract in AD, suggesting that disruption in the white matter tracts near the temporal lobe may represent the secondary consequence of the medial temporal lobe pathology in AD. Moreover, the regionally-specific patterns of FA and shape of the white matter tracts were shown to be of sufficient sensitivity to robustly differentiate patients with AD from healthy comparison controls when compared with the mean FA and volumes within the regions of the white matter tracts. Finally, greater FA or deformation abnormalities of the white matter tracts were associated with lower MMSE scores.

Conclusion

The regionally-specific shape and FA patterns could be potential imaging markers for differentiating AD from normal aging.